Pseudorabies virus induces ferroptosis by disrupting iron homeostasis through activation of TfR1 and ferritinophagy

To preliminarily investigate the relationship between PRV infection and PCD, we determined the key indicators of apoptosis, necroptosis, and pyroptosis. The results showed that increasing the PRV infection dose led to a gradual increase in apoptosis levels (). Further analysis revealed a slight increase in p-MLKL and the presence of mildly cleaved Gasdermin D (GSDMD) at a high MOI (multiplicity of infection), although these changes were not significant, and no cleaved form of Gasdermin E (GSDME) was observed (). Fig. S1A Fig. S1B

Ferroptosis, a recently identified iron-dependent form of PCD, plays a critical role in viral replication and pathogenesis (17). To determine whether PRV infection induces ferroptosis, we analyzed mitochondrial morphology in mock-infected and PRV-infected N2a cells using transmission electron microscopy. PRV-infected cells exhibited swollen mitochondria with reduced cristae compared to uninfected cells (Fig. 1A) and showed significantly smaller mitochondrial diameters (Fig. 1B). Next, we treated cells with increasing doses of PRV and observed a corresponding decrease in cell viability, which was rescued by Ferrostatin-1 (Fer-1), a specific ferroptosis inhibitor (33, 34) (Fig. 1C). As a positive control, we used RSL3, a known ferroptosis inducer, and found that PRV infection significantly increased levels of lactate dehydrogenase (LDH) release, malondialdehyde (MDA), Fe²⁺, and ROS (Fig. 1D, E, G and H). To assess lipid peroxidation, we used the fluorescent probe C11-BODIPY 581/591. In its reduced form, C11-BODIPY emits red fluorescence, which shifts to green upon oxidation, enabling real-time detection of lipid peroxidation. Our results showed that PRV infection induced dose-dependent lipid peroxidation (Fig. 1F). Similar results were observed in two additional cell lines: PK-15 (Fig. S2A through H) and SH-SY5Y cells (Fig. S2I through P). In contrast, PRV inactivated by ultraviolet irradiation (PRV-UV) failed to induce ferroptosis (Fig. S3A through G). Together, these findings demonstrate that PRV infection can induce ferroptosis.

Given the emerging nature of ferroptosis, its role in PRV infection was subsequently determined. To investigate the role of ferroptosis in PRV replication, N2a cells were treated with varying concentrations of Ferrostatin-1 (Fer-1), a specific ferroptosis inhibitor, followed by PRV infection. Importantly, Fer-1 treatment at the tested doses did not affect cell viability under normal conditions (Fig. 2A). However, under PRV infection, Fer-1 significantly increased cell viability in a dose-dependent manner compared to the vehicle control (DMSO) (Fig. 2B). Correspondingly, viral titers were reduced in a dose-dependent manner following Fer-1 treatment (Fig. 2C), and expression of the viral protein gB gradually declined with increasing Fer-1 concentrations (Fig. 2D). Similar results were observed in PK-15 (Fig. S4A through D) and SH-SY5Y cells (Fig. S4E through H). We also examined the effect of Fer-1 on apoptosis. Flow cytometry analysis revealed that although apoptosis levels increased over time in both treatment groups during PRV infection, cells treated with Fer-1 exhibited a significantly greater reduction in apoptosis compared to the DMSO control group, particularly at later time points (Fig. 2E). These findings suggest that the ferroptosis inhibitor Fer-1 can suppress both PRV-induced cell death and viral replication, indicating that PRV-induced ferroptosis contributes to efficient viral replication.

We observed that the concentration of Fe²⁺ increased in a dose-dependent manner following PRV infection (Fig. 1G). To determine whether iron concentration influences PRV-induced ferroptosis and viral replication, we utilized ferric ammonium citrate (FAC), a non-transferrin-bound iron compound widely used to elevate extracellular iron levels (35, 36). N2a cells were treated with either FAC or vehicle (DMSO) and subsequently infected with PRV. FAC treatment had no significant impact on cell viability (Fig. 3A). To further elucidate the role of ferroptosis in PRV infection, we assessed key ferroptosis markers, including lipid peroxidation, Fe²⁺ levels, and ROS. In mock-infected N2a cells, FAC treatment did not significantly alter any of these markers compared to the DMSO control group. However, in PRV-infected cells, treated with FAC, lipid peroxidation, Fe²⁺, and ROS were significantly elevated relative to the control (DMSO) (Fig. 3B through D). Moreover, FAC treatment significantly increased both viral titers and expression of the viral envelope protein gB (Fig. 3E and F). To complement these findings, we performed parallel experiments using deferoxamine mesylate (DFOM), an iron chelator that reduces extracellular iron levels by forming stable iron complexes (37, 38). DFOM treatment did not affect cell viability at the concentrations used (Fig. 3G). However, it significantly reduced lipid peroxidation, Fe²⁺ levels, and ROS production, as well as viral titers and gB expression (Fig. 3H through L). Collectively, these results indicate that PRV-induced ferroptosis and viral replication are positively correlated with extracellular iron concentration.

Since iron availability is critical for PRV-induced ferroptosis, we next investigated the relationship between PRV infection and iron metabolism. TfR1 is one of the major proteins mediating iron entry into cells and plays a key role in regulating cellular iron metabolism and maintaining iron balance (39). FTH1, a major subunit of ferritin, the primary intracellular iron storage complex, stores approximately 75% of newly imported iron and protects cells from iron overload and ferroptosis (40, 41). Degradation of FTH1 leads to the release of Fe²⁺, contributing to oxidative stress and ferroptosis (42). To determine whether PRV affects these pathways, we measured TfR1 and FTH1 expression following PRV infection. We observed that PRV infection significantly upregulated TfR1 expression while downregulating FTH1 expression (Fig. 4A). These alterations are consistent with the induction of ferroptosis and suggest a disruption of iron homeostasis during PRV infection.

To further explore the roles of TfR1 and FTH1 in PRV-induced ferroptosis, we knocked down TfR1 using short hairpin RNA (shRNA) and overexpressed FTH1 via lentiviral transduction in N2a cells. Knockdown and overexpression efficiencies were confirmed by RT-qPCR and western blot analysis (). Fig. S5

We then assessed cell viability, lipid peroxidation, Fe²⁺ levels, and ROS in PRV-infected wild-type and TfR1-knockdown cells. Ferrostatin-1 (Fer-1) effectively inhibited PRV-induced ferroptosis, and TfR1 knockdown significantly reduced ferroptosis-mediated cell death in infected cells (Fig. 4B). Correspondingly, TfR1-deficient cells exhibited reduced lipid peroxidation, Fe²⁺ levels, and ROS generation compared to wild-type controls (Fig. 4C through E). Importantly, the replication of PRV was significantly reduced, as evidenced by reduced viral titers and expression of viral protein gB (Fig. 4F and G). Parallel experiments in FTH1-overexpressing N2a cells showed similar protective effects. Upon PRV infection, FTH1-overexpressing cells exhibited decreased cell death, lipid peroxidation, Fe²⁺ accumulation, ROS production, and viral replication compared to wild-type cells (Fig. 4H through M).

Collectively, these findings demonstrate that PRV infection disrupts iron homeostasis through two complementary mechanisms: it upregulates TfR1 to enhance iron influx and downregulates FTH1 to promote intracellular Fe²⁺ release. These changes synergistically increase the labile iron pool, thereby facilitating ferroptosis and viral replication.

Next, we investigated the mechanism by which PRV infection upregulates TfR1 expression. TfR1 is regulated at the transcriptional, post-transcriptional, and post-translational levels (43). To determine whether PRV affects TfR1 transcription, we performed RT-qPCR and western blot analysis. We found that both TfR1 mRNA and protein levels were significantly increased upon PRV infection (Fig. 5A). Transcription of TfR1 is regulated by hypoxia-inducible factors (HIFs), which bind to hypoxia response elements (HREs) in the TfR1 promoter (43, 44). Post-transcriptionally, iron regulatory proteins (IRP1 and IRP2) stabilize TfR1 mRNA by binding to iron-responsive elements (IREs) in the 3′ untranslated region (3′-UTR), thereby preventing Regnase-1 (Reg-1)-mediated mRNA degradation (45). To assess the potential involvement of these pathways, we examined the expression of HIF-1α, HIF-1β, IRP1, and IRP2 at various time points following PRV infection. The levels of HIF-1α and HIF-1β increased over time, whereas IRP1 and IRP2 expression remained unchanged (Fig. 5B). To clarify their roles in PRV-mediated regulation of TfR1, we knocked down HIF-1α, HIF-1β, IRP1, and IRP2 in N2a cells. Knockdown efficiency was confirmed by RT-qPCR and western blot (Fig. S6). We then compared TfR1 mRNA and protein expression between knockdown and wild-type cells following PRV infection. Notably, HIF-1β knockdown led to a marked reduction in TfR1 mRNA and protein levels, while knockdown of HIF-1α, IRP1, or IRP2 had no significant effect (Fig. 5C through F). These results suggest that PRV induces TfR1 transcription through a HIF-1-dependent mechanism, with the HIF-1β subunit playing a critical role.

To further validate this hypothesis, we assessed cell viability, ferroptosis markers, and viral replication in HIF-1β-knockdown and wild-type N2a cells following PRV infection. HIF-1β knockdown significantly reduced cell death, lipid peroxidation, Fe²⁺ accumulation, ROS production (Fig. 5G through J), and viral replication (Fig. 5K and L). Collectively, these findings indicate that PRV promotes TfR1 expression via HIF-1β, leading to enhanced iron uptake and increased susceptibility to ferroptosis.

TfR1 is a membrane protein that requires transport to the cell surface to perform its biological function, a process mediated by Rab11a (46, 47). To examine whether PRV affects Rab11a expression, we performed western blot analysis before and after PRV infection. The results showed a significant increase in Rab11a expression following infection (Fig. 6A). Moreover, co-immunoprecipitation and proximity ligation assay (PLA) experiments revealed that the interaction between TfR1 and Rab11a was enhanced upon PRV infection (Fig. 6B and C), likely due to the upregulation of both TfR1 and Rab11a expression in response to the virus.

To further assess the role of Rab11a in TfR1 trafficking during PRV infection, we knocked down Rab11a in N2a cells. Knockdown efficiency was confirmed by RT-qPCR and western blot analysis (Fig. S7A). Confocal microscopy revealed increased localization of TfR1 at the cell membrane following PRV-GFP infection, which was markedly reduced in Rab11a-knockdown cells (Fig. 6D). To test the functional consequences of Rab11a depletion, Rab11a-knockdown and wild-type N2a cells were infected with PRV, and subsequent ferroptosis and viral replication were evaluated. Rab11a knockdown led to reduced levels of cell death, lipid peroxidation, Fe²⁺ accumulation, and ROS production, as well as diminished PRV replication (Fig. 6E through J). Together, these findings suggest that PRV infection promotes Rab11a-dependent translocation of TfR1 to the cell membrane, enabling TfR1 to perform its function and thereby increasing cellular susceptibility to ferroptosis.

Next, we investigated the mechanism underlying FTH1 degradation. Typically, FTH1 degradation occurs through ferritinophagy (9). Therefore, we sought to elucidate the relationship between PRV infection and ferritinophagy. Ferritinophagy is a selective autophagy process mediated by the cargo receptor NCOA4, which binds ferritin and directs it to the autolysosome, playing a critical role in maintaining iron homeostasis. Additionally, the selective autophagy receptor TAX1BP1 directly interacts with NCOA4, facilitating ferritin transport to lysosomes for degradation (48). We analyzed the expression levels of NCOA4 and TAX1BP1 by western blot and observed that PRV infection induced concomitant degradation of NCOA4, TAX1BP1, and FTH1 in N2a cells, suggesting a specific role for these proteins in PRV-induced ferritin degradation (Fig. 7A). To confirm whether this process is autophagy-dependent, we treated cells with the autophagy inhibitor 3-MA. Without 3-MA treatment, NCOA4, TAX1BP1, and FTH1 were degraded following PRV infection; however, 3-MA treatment inhibited their degradation (Fig. 7B). During ferritinophagy, FTH1 is translocated to lysosomes, which are marked by LAMP1. We observed increased co-localization of FTH1 with the lysosomal marker LAMP1 after PRV-GFP infection by using confocal microscopy (Fig. 7C). We further examined the interaction between NCOA4, TAX1BP1, and FTH1 by co-immunoprecipitation. Our results showed that PRV infection enhanced the interaction among these proteins, leading to FTH1 degradation via ferritinophagy (Fig. 7D).

To investigate the roles of NCOA4 and TAX1BP1, we knocked down these proteins in N2a cells, with knockdown efficiency confirmed by RT-qPCR and western blot (Fig. S7B and C). In knockdown cells, FTH1 degradation was significantly reduced compared to controls (Fig. 7E and F), demonstrating that PRV-induced ferritinophagy depends on NCOA4 and TAX1BP1. Next, we infected NCOA4- and TAX1BP1-knockdown cells and wild-type cells with PRV to assess ferroptosis and viral replication. Knockdown of either NCOA4 or TAX1BP1 resulted in decreased ferroptosis markers (Fig. 7G through J and M through P) and reduced PRV replication (Fig. 7K, L, Q and R). Together, these findings indicate that PRV infection promotes the interaction of TAX1BP1, NCOA4, and FTH1, activating ferritinophagy and thereby increasing intracellular ferrous iron levels through ferritin degradation.

Human embryonic kidney-293T (HEK-293T, CRL-11268), Porcine kidney cell 15 (PK-15; CCL-33), Vero (CCL-81), SH-SY5Y (CRL-2266), and Neuro-2a (N2a; CCL-131) cells were obtained from the American Type Culture Collection (ATCC). PRV ZJ01 (GenBank: KM061380.1) is a prototype PRV strain used in this study as wild-type PRV (59). The recombinant virus PRV-GFP was constructed by inserting a GFP under the control of cytomegalovirus promoter into the genome of PRV ZJ01 by using the homogenous recombination. Preparation of viral stock and titration of infectivity were prepared using Vero cells.

The following primary antibodies were used: mouse anti-PRV-gB protein (produced in our laboratory), mouse anti-PRV-VP16 protein (produced in our laboratory), mouse anti-β-actin (Sigma-Aldrich, USA; no. A5441), mouse anti-TfR1 (Cell Signaling Technology, USA; no. 46222), mouse anti-TfR1 (Sigma-Aldrich, USA; no. MABC1765), rabbit anti-FTH1 (Cell Signaling Technology, USA; no. 3998), rabbit anti-IRP1 (Cell Signaling Technology, USA; no. 20272), rabbit anti-IRP2 (Cell Signaling Technology, USA; no. 37135), rabbit anti-HIF-1α (Cell Signaling Technology, USA; no. 14179), rabbit anti-HIF-1β (Cell Signaling Technology, USA; no. 5537), rabbit anti-Rab11a (Proteintech, USA; no. 20229-1-AP), mouse anti-NCOA4 (Santa Cruz Biotechnology, USA; no. sc-373739), rabbit anti-LAMP1 (Cell Signaling Technology, USA; no. 9091), rabbit anti-TAX1BP1 (Proteintech, USA; no. 14424-1-AP), rabbit anti-GSDME (Abcam, USA; no. ab215191), rabbit anti-GSDMD (Novus Biologicals, USA; no. NBP2-33422), rabbit anti-MLKL (Cell Signaling Technology, USA; no. 37705), and rabbit anti-p-MLKL (Affinity Biosciences, China; no. AF7420). Horseradish peroxidase (HRP)-labeled rabbit (Cat No. A0208) or mouse (Cat No. A0216) secondary antibodies were purchased from Beyotime (China). The following chemicals were used: RSL3 (Selleck, USA; no. S8155), Fer-1 (Selleck, USA; no. S7243), FAC (Selleck, USA; no. E0375), DFOM (Selleck, USA; no. S5742).

The FTH1 gene (NCBI Reference Sequence: NM_010239.2↗) was amplified from N2a cells (Mus musculus) by RT-PCR, the primers sequences are listed in Table S1. The amplified FTH1 fragment was digested by BamHI (Thermo Fisher, USA) and EcoRI (Thermo Fisher, USA) and then cloned into pCMV-N-FLAG (4,321, Beyotime, no. D2722), resulting in pCMV-FLAG-FTH1. The pLKO.1-TRC and lentivirus package plasmids pVSV-G (8,454, deposited by Bob Weinberg) and psPAX2 (12,260, deposited by Didier Trono) were purchased from Addgene. The shRNA for shTfR1, shFTH1, shHIF-1α, shHIF-1β, shIRP1, shIRP2, shRab11a, shNCOA4, and shTAX1BP1 was cloned into pLKO.1-TRC, resulting in pLKO.1-shTfR1, pLKO.1-shFTH1, pLKO.1-shHIF-1α, pLKO.1-shHIF-1β, pLKO.1-shIRP1, pLKO.1-shIRP2, pLKO.1-shRab11a, pLKO.1-shNCOA4, and pLKO.1-shTAX1BP1, respectively. The recombinant plasmid sequences were confirmed by DNA Sanger sequencing and then transfected into N2a cells or HEK-293T cells using Lipofectamine 3000 (Thermo Fisher, USA) according to the manufacturer's instructions. For gene knockdown experiments, pLKO.1-shTfR1, pLKO.1-shFTH1, pLKO.1-shHIF-1α, pLKO.1-shHIF-1β, pLKO.1-shIRP1, pLKO.1-shIRP2, pLKO.1-shRab11a, pLKO.1-shNCOA4, and pLKO.1-shTAX1BP1 were co-transfected with lentivirus package plasmids (pVSV-G and psPAX2) into HEK-293T cells to generate lentiviruses. Subsequently, these lentiviruses were isolated and used to infect N2a cells. At 36 hpi (hours post infection), infected cells were cultured with 5 µg/mL puromycin to select knockdown cells. The efficiency of knockdown was assessed by RT-qPCR and western blot. The shRNA sequences are listed in Table S1.

N2a, PK-15, and SH-SY5Y cells were harvested and fixed with 2.5% glutaraldehyde at 4°C for 12 h. Next, they were treated with 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Finally, sections were observed using a Hitachi TEM HT7700 (Hitachi, Tokyo, Japan). The diameters of mitochondria were measured by using ImageJ 1.8.0 software (National Institutes of Health).

The levels of cell viability were determined using cell counting kit 8 (CCK-8) (Beyotime, China, no. C0038) according to the manufacturer's instructions. Briefly, N2a, PK-15, and SH-SY5Y cells were seeded into 96-well plates and treated with different chemicals (RSL3, Fer-1, FAC, and DFOM) or PRV for 24 h. Then, 10 µL CCK-8 solution was added to each well. After incubating for 1 h, absorbance was measured at a wavelength of 450 nm by using microplate spectrophotometer (Agilent BioTek, USA).

The levels of cytotoxicity were determined using LDH Cytotoxicity Assay kit (Beyotime, China, no. C0016) according to the manufacturer's instructions. Briefly, N2a, PK-15, and SH-SY5Y cells were seeded into 96-well plates and treated with RSL3 or PRV for 24 h. Then, 120 µL supernatant of each well was transferred into new 96-well plates. Sixty microliters of LDH working solution was added to each well. After incubating for 30 min, absorbance of each well was measured at a wavelength of 490 nm by using microplate spectrophotometer (Agilent BioTek, USA).

Malondialdehyde (MDA) is an end-product of lipid peroxidation. The levels of MDA were determined using the Lipid Peroxidation MDA Assay Kit (Beyotime, China, S0131S) according to the manufacturer's instructions. Briefly, N2a, PK-15, and SH-SY5Y cells were seeded into 6-well plates and treated with different chemicals (RSL3, FAC, and DFOM) or PRV for 24 h. Next, cells were lysed, and 100 µL of cell lysates from each well was mixed with 200 µL TBA working solution. After incubating for 15 min at 100°C, absorbance was measured at a wavelength of 532 nm by using microplate spectrophotometer (Agilent BioTek, USA).

The level of lipid peroxidation was determined using C11-BODIPY 581/591 (Thermo Fisher, USA, D3861) according to the manufacturer's instructions. C11-BODIPY 581/591 was diluted to 10 mM with DMSO as a stock solution. N2a cells were seeded into a 6-well plate and treated with different titers of PRV for 24 h. Next, the C11-BODIPY 581/591 stock solution was diluted with DMEM at a 1:1,000 ratio to achieve a final concentration of 10 µM. The cell culture medium was then removed, and 1 mL of the diluted C11-BODIPY 581/591 solution was added to each well of the 6-well plate. Cells were incubated at 37°C for 30 min, followed by three washes with DMEM. Finally, cells were collected and analyzed by flow cytometry (Beckman Coulter, USA).

The levels of ferrous iron were determined using an iron assay kit (Sigma-Aldrich, USA, no. MAK025) according to the manufacturer's instructions. Briefly, N2a, PK-15, and SH-SY5Y cells were seeded into 6-well plates and treated with different chemicals (RSL3, FAC, and DFOM) or PRV for 24 h. Then, the cells were lysed, and 80 µL cell lysates of each well was mixed with 80 µL working solution. After incubating for 30 min, absorbance of each well was measured at a wavelength of 490 nm by using microplate spectrophotometer (Agilent BioTek, USA).

The levels of reactive oxygen species were determined using ROS Assay Kit (Beyotime, China, no. S0033) according to the manufacturer's instructions. Briefly, N2a, PK-15, and SH-SY5Y cells were seeded into 6-well plates and treated with different chemicals (RSL3, FAC, and DFOM) or PRV for 24 h. Next, DCFH-DA has been diluted with DMEM at 1:1,000 to give a final concentration of 10 µM. Removed the cell culture medium and added 1 mL of diluted DCFH-DA for one well of a six-well plate. The cells were incubated in a cell incubator at 37°C for 20 min and then washed three times with DMEM. Cells were collected and analyzed by flow cytometry (Beckman Coulter, USA).

Total cell proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, and leupeptin). Protein concentrations were determined using bicinchoninic acid (BCA) assay according to the manufacturer's instructions (Beyotime, China). After protein boiling and denaturation, an aliquot of proteins was separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) film (EMD Millipore, USA). The membrane was blocked for 2 h at room temperature with 5% skim milk in TBS-T (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20). Primary antibodies were added to the membrane, which was then incubated at 4°C overnight. The membrane was washed three times for 10 min with TBS-T and incubated with HRP-labeled anti-rabbit or anti-mouse antibodies for 1 h at room temperature. The membrane was washed three times for 10 min with TBS-T, and the bands were visualized using enhanced chemiluminescence (ECL) reagent and a gel imaging system (ImageQuant LAS 500; Cytiva). Finally, bands were quantitated using ImageJ 1.8.0 software (National Institutes of Health).

Rabbit anti-TfR1, mouse anti-NCOA4, or normal IgG were mixed with Protein A + G Magnetic Beads (Beyotime, China, P2108) and incubated at room temperature for 1 h. Beads were washed three times with PBS (155.2 mM NaCl, 1.059 mM KH2PO4, 2.967 mM Na2HPO4). Cells were harvested and lysed with NP-40 buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1:400 protease inhibitor mixture). Supernatants were collected and mixed with pretreatment magnetic beads and incubated at 4°C for 6 h. Beads were washed three times with washing buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, and protease inhibitor mixture) and collected. The precipitated proteins were analyzed by western blot using the indicated antibodies.

The FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, USA; no. 556547) was used to detect early stages of cell death. Briefly, cells were washed twice with PBS and resuspended in 1× binding buffer at a concentration of 1 × 10⁶ cells/mL. To 100 µL of the mixture (~10⁵ cells), 5 µL of FITC Annexin V and 5 µL of propidium iodide (50 µg/mL stock) were added, and the cells were incubated for 15 min at room temperature in the dark. After incubation, 400 µL of 1× binding buffer was added to each tube, and the cells were analyzed by flow cytometry. Three controls were included for flow cytometry analysis: (i) unstained cells, (ii) cells with FITC Annexin V but without PI, and (iii) cells without FITC Annexin V but with PI.

Cells were either mock- or PRV-infected (MOI = 1) at various time points. PLA was performed using the Duolink In Situ Detection Reagents Green (Sigma-Aldrich, USA, no. DUO92014). Following the manufacturer's instructions, cells were first subjected to fixation, recovery, and permeabilization. After blocking with Duolink blocking buffer at 37°C for 1 h, cells were incubated with mouse anti-TfR1 antibody and rabbit anti-Rab11a antibody at 37°C for 2 h. The cells were then treated with pre-diluted anti-rabbit and anti-mouse minus probes at 37°C for 1 h. Subsequently, the cells were incubated consecutively with 1× ligase and 1× polymerase for 30 and 100 min, respectively, and finally mounted on slides with Duolink In Situ Mounting Medium containing DAPI.

Cells were harvested, and total RNA was extracted from PRV-infected or mock-infect cells using the Total RNA Kit I (Omega Bio-tek, USA, no. R6834). RNA was then reverse transcribed using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China, no. R211) according to the manufacturer's instructions. RT-qPCR was performed with the AceQ qPCR SYBR Green Master Mix (Vazyme, China, no. Q112). The RT-qPCR primer sequences are listed in. Table S1

Cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4°C. After three washes with ice-cold PBS, cells were permeabilized with 0.1% Triton X-100 for 15 min and then blocked in 5% bovine serum albumin (BSA) in PBS for 1 h at 37°C. Cells were incubated with indicated primary antibodies (rabbit anti-TfR1, rabbit anti-LAMP1, or mouse anti-Flag) for 1 h at 37°C. Cells were washed with PBS and incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L) or Alexa Fluor 647-conjugated goat anti-mouse IgG (H + L) (Thermo Fisher, USA, no. A-11012/A-21235) for 1 h at 37°C in the dark and then stained with DAPI.

All data were analyzed using GraphPad Prism 7.0 software (GraphPad Software, Inc.) and are presented as mean ± SD. Western blot band intensities were quantified using ImageJ 1.8.0 software (National Institutes of Health) and normalized to β-actin. Statistical comparisons were made using t-tests. Unless otherwise noted in the legends, P-values were calculated from three biological replicates. Data were reproduced in independent experiments as described in the legends.