To enhance the efficacy of Newcastle disease virus (NDV)-vectored bivalent vaccines against novel variant infectious bursal disease virus (nVarIBDV), we engineered recombinant NDV strains expressing nVarIBDV VP2 proteins fused with the viral nucleocapsid NC motif, thereby leveraging the NDV assembly pathway. The resulting vaccine candidates, rDM-VP2NC and rDM-VP2mNC, exhibited markedly enhanced VP2 expression in vitro. Furthermore, the NC motif promoted VP2 co‑localization with the NDV M protein near the cell membrane and facilitated its efficient incorporation into budding virus-like particles (VLPs) and virions. In chickens challenged with a nVarIBDV strain, immunization with rDM‑VP2NC or rDM‑VP2mNC provided superior protection compared to the parental rDM‑VP2 strain, as demonstrated by a drastic reduction in bursal viral load and the absence of detectable pathological lesions. These results establish the NC motif as a key determinant for enhancing antigen assembly and immunogenicity in NDV vectors. Collectively, this work presents a novel strategy for engineering NDV as a vector that directly packages protein cargo within virions, with broad implications for vaccine and oncolytic virotherapy development.

DEAD/H-box RNA helicases are critical regulators of host antiviral innate immunity. In this study, we utilized an RNA-binding protein knockout sub-library to identify DDX46, a member of the DEAD/H-box RNA helicase family, as an essential proviral host factor for RNA virus replication. While DDX46 has been shown to sequester demethylated innate immune transcripts in the nucleus and dampen interferon (IFN) production, the mechanisms underlying its regulation during viral infection remain unclear. Here, we report that RNA virus infection induces caspase-dependent cleavage of DDX46, triggering its translocation from the nucleus to the cytoplasm. This translocation unchains innate immune transcripts from nuclear retention, licensing their rapid translation and potentiating robust IFN responses. Our findings reveal a novel regulatory mechanism by which post-translational modification and subcellular relocalization of DDX46 fine-tune the host antiviral response, highlighting the functional versatility of RNA helicases in host-virus interactions.IMPORTANCEUnderstanding how host cells regulate innate immune responses to viral infection is essential for developing effective antiviral strategies. Our study uncovers a critical role for caspase-dependent cleavage and nuclear-cytoplasmic translocation of DDX46 in promoting antiviral innate immunity. These findings not only expand our knowledge of the dynamic regulation of DEAD/H-box RNA helicases during infection but also suggest that targeting the post-translational modification and localization of such helicases may offer new avenues for antiviral therapeutic development.

[This corrects the article DOI: 10.1371/journal.ppat.1012987.].

Newcastle disease virus (NDV) is a highly lethal and contagious viral pathogen, and it is also a potent oncolytic virus that selectively replicates in tumor cells. NDV demonstrates high replication efficiency in avian and tumor cells, causing various types of cell death, including ferroptosis, necrosis, apoptosis and autophagic cell death, with apoptosis being the most thoroughly studied. Organelles play critical and distinctive roles in the regulation and execution of apoptosis. However, the involvement of peroxisomes, an important organelle that regulates redox balance and lipid biosynthesis, in virus-induced apoptosis remains unclear. Our findings reveal that NDV infection promotes the downregulation of several peroxisome biogenesis factors (PEXs) at the mRNA level. Peroxisomal biogenesis factor 5 (PEX5), a critical peroxisomal shuttle protein, was identified to be significantly downregulated at both the mRNA and protein levels. Further, gain- and loss-of-function experiments demonstrated the negative regulation of NDV-induced apoptosis by PEX5. In addition, PEX5 inhibits NDV-induced apoptosis by regulating the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) expression. These findings reveal a novel mechanism by which NDV-induced apoptosis is modulated through the downregulation of PEXs, particularly PEX5, shedding light on the potential role of peroxisome in apoptosis regulation in response to virus infection.

Marek's disease (MD) is a highly contagious lymphoproliferative disease caused by Marek's disease virus (MDV), typically characterized by severe immunosuppression. In this study, exosomes were isolated and purified from chicken embryo fibroblasts (CEFs) infected with the virulent MDV strain Md5 (Exo-Md5) and uninfected controls (Exo-NC), respectively. Combining small RNA sequencing, mass spectrometry, and Western blot analysis, we confirmed that MDV infection significantly remodels the exosomal cargo composition. Exo-Md5 exhibited a predominant downregulation of miRNAs. Subsequent GO and KEGG enrichment analyses revealed that these downregulated miRNAs were significantly enriched in critical pathways closely associated with host immunity and viral replication, including T cell receptor signaling, Toll-like receptor signaling, type I interferon response, and cell cycle regulation. Proteomic analysis further demonstrated that MDV infection resulted in 147 upregulated proteins in exosomes. Integrated enrichment analysis using EggNOG, KEGG, and GO databases showed that differentially expressed proteins were predominantly enriched in pathways associated with intracellular transport, metabolic regulation, immune modulation, organismal homeostasis, and tumor-related processes. Additionally, 20 MDV-derived components were identified in Exo-Md5, including the tegument protein VP22 mediating intercellular spread, the envelope glycoprotein gB involved in viral entry, and the phosphoprotein pp38 associated with cell transformation, indicating that MDV-infected cells can package viral proteins into exosomes through sorting mechanisms. Functional assays further demonstrated that exosomes derived from Md5-infected cells (Exo-Md5) were able to reduce the proportion of CD4+ T lymphocytes and promote viral replication, while inhibition of exosome secretion suppressed viral propagation. Collectively, the findings of this study provide novel experimental evidence for elucidating the molecular mechanisms underlying MDV-mediated immunosuppression, immune evasion, and efficient replication.

Background Newcastle disease virus (NDV), a significant avian pathogen and promising oncolytic agent, relies on host metabolic pathways for replication. However, the metabolic alterations induced by NDV, particularly the connections at the gene and protein levels, remain poorly characterized. Results This study employed integrated transcriptomic, proteomic, and non-targeted metabolomic analyses to delineate the global metabolic changes in NDV-infected A549 cells. We identified 8,101 differentially expressed genes (DEGs), 1,587 differentially expressed proteins (DEPs), and 257 differentially expressed metabolites (DEMs) associated with organelle function, innate immunity, and metabolism. Crucially, our multi-omics approach revealed that NDV significantly remodels glycerophospholipid metabolism. NDV depleted Lysophosphatidylcholine (LPC) and Lysophosphatidylethanolamine (LPE), as well as specific phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species, while increasing phosphatidylserine (PS) at the late stage of infection. Strikingly, exogenous supplementation of unsaturated fatty acids, choline, phosphorylcholine, ethanolamine, phosphatidylethanolamine, and inositol markedly enhanced NDV replication. Concomitantly, NDV infection upregulated the transcriptional levels of key enzymes involved in glycerophospholipid biosynthesis. Conclusions This study demonstrates for the first time that NDV actively reprograms host glycerophospholipid metabolism to facilitate viral replication. This study uncovers a novel mechanism of NDV-host interaction and provides crucial insights for oncolytic strategies targeting this metabolic vulnerability.

Lipid droplets (LDs) are dynamic organelles that store neutral lipids and maintain lipid homeostasis. Many viruses exploit LDs as replication platforms or lipid sources, but their role in supplying membrane lipids for viral assembly remains unclear. Newcastle disease virus (NDV), an enveloped RNA virus with oncolytic potential, extensively remodels host metabolism, yet its impact on LD lipid mobilization is unknown. Here, we show that NDV reprograms host lipid metabolism via SQSTM1/p62-dependent lipophagy, selectively degrading triglycerides (TAGs) enriched in unsaturated fatty acids (UFAs). Lipidomics revealed concurrent depletion of UFA-containing triglycerides (UFA-TAGs) and UFA-containing phosphatidylcholines (UFA-PCs) during infection. Inhibition of lipophagy blocked LD degradation, reduced viral replication, and suppressed UFA-PC formation. Isotope tracing demonstrated that lipophagy-derived UFAs are incorporated into phosphatidylcholines (PCs) via the Kennedy pathway, whereas β-oxidation was dispensable. UFA supplementation rescued viral replication under lipophagy blockade and promoted virus-like particle (VLP) release, indicating that UFA-PCs facilitate viral budding. These findings uncover a distinct NDV strategy linking lipophagy-driven UFA release to phospholipid synthesis and membrane remodeling, revealing a lipid-based metabolic vulnerability for antiviral and oncolytic interventions.Abbreviations: AP: autophagosome; ATG: autophagy related; ATP: adenosine triphosphate; CQ: chloroquine; EGFP: enhance green fluorescent protein; FFA: free fatty acid; HN: Hemagglutinin-Neuraminidase; LA: linoleic acid; LD: lipid droplet; LIPA: lipase A, lysosomal acid type; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NDV: newcastle disease virus; NP: nucleoprotein; OA: oleic acid; PA: palmitic acid; PC: phosphatidylcholine; PLIN2/ADRP: perilipin 2; PNPLA2/ATGL: patatin like phospholipase domain containing 2; POA: palmitoleic acid; SFA: saturated fatty acid; TAG: triglyceride; UFA: unsaturated fatty acid; UFA-PC: UFA-containing phosphatidylcholine; VLP: virus-like particle.

Mitochondria serve as the cellular "power plants," supplying energy and regulating metabolism, signal transduction, and other physiological processes. To successfully replicate within host cells, viruses have evolved multiple strategies to hijack mitochondrial functions. The oncolytic Newcastle disease virus (NDV) causes severe organelle damage in tumor cells; however, how it manipulates mitochondrial architecture to facilitate its own replication remains poorly understood. Here, we provide evidence that NDV infection disrupts mitochondrial spatial distribution and imbalances mitochondrial fusion and fission, leading to mitochondrial structural damage. The resulting accumulation of fragmented mitochondria is cleared via PRKN-dependent mitophagy, a process that supports NDV replication. Interestingly, although MAVS (mitochondrial antiviral signaling protein) is degraded along with mitophagy, genetic ablation of PRKN - while blocking MAVS degradation - does not restore downstream innate immune responses. This indicates that NDV exploits mitophagy to enhance replication through mechanisms not entirely dependent on the suppression of MAVS-mediated immunity. Given the central role of mitochondria, we further explored the link between amino acid metabolism and viral proliferation after NDV infection. Our results show that NDV-induced mitophagy leads to the accumulation of free amino acids in host cells, and this metabolic reprogramming promotes viral replication. In summary, we show that NDV drives its replication by remodeling mitochondrial dynamics to induce mitophagy, which in turn triggers an amino acid metabolic reprogramming that benefits the virus. This provides new insights into the mechanisms supporting efficient oncolytic NDV replication, offering potential avenues for therapeutic intervention in oncolytic virus therapy.Abbreviations: CCCP: carbonyl cyanide m-chlorophenylhydrazone; COX4/COX IV: cytochrome c oxidase subunit 4; CQ: chloroquine; DENV: dengue virus; DNM1L/DRP1: dynamin 1 lik;ETC: electron transport chain; FIS1: fission, mitochondrial 1; HBV: hepatitis B virus; IAV: influenza A virus; IMM: inner mitochondrial membrane; JEV: japanese encephalitis virus; MAVS: mitochondrial antiviral signaling protein; MFF: mitochondrial fission factor; MFN1: mitofusin 1; MFN2: mitofusin 2; MOI: multiplicity of infection; MV: measles virus; NDV: Newcastle disease virus; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; PINK1: PTEN induced kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; RLR: RIG-I-like receptor; SDHA: succinate dehydrogenase complex flavoprotein subunit A; TCA: tricarboxylic acid cycle; TCID50: tissue culture infective doses; TEM: transmission electron microscopy; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20.

Oncolytic viruses selectively infect and kill tumor cells, but the metabolic adaptations that support their replication remain incompletely understood. Here, using oncolytic Newcastle disease virus (NDV) as a model, we identify glutamic-oxaloacetic transaminase 1 (GOT1) as a key metabolic enzyme required for efficient viral replication through its dual role in de novo pyrimidine synthesis. In NDV-infected tumor cells, GOT1 promotes aspartate production through the malate-aspartate shuttle to support pyrimidine biosynthesis, while also maintaining NAD+/NADH homeostasis to activate the mTOR-S6K-CAD signaling axis and further enhance pyrimidine synthesis. These GOT1-dependent metabolic and signaling adaptations sustain pyrimidine biosynthesis and viral replication. In addition, NDV infection promotes pyrimidinosome assembly, and GOT1 functions as a pyrimidinosome-associated component. Together, these findings reveal a mechanism by which oncolytic NDV rewires host pyrimidine metabolism to support its replication and provide a rationale for metabolic modulation of oncolytic virotherapy.

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs widely expressed across diverse organisms. These molecules are involved in various biological processes, such as transcriptional and post-transcriptional regulation, protein scaffolding, and acting as miRNA sponges. Newcastle disease virus (NDV) replicates and proliferates in a variety of cells, inducing severe organelle stress damage, autophagy, and even cell death. Previous studies have reported that NDV hijacks and uses autophagy to promote its replication and proliferation. However, the involvement and regulatory mechanisms of circRNAs in this process remain largely unexplored. The present study aimed to identify novel circRNAs that modulate NDV replication through autophagy and elucidate their underlying mechanisms. Our findings revealed that circRNA0857 was significantly upregulated in NDV-infected cells. Functional assays showed that silencing circRNA0857 markedly inhibited NDV replication, while its overexpression enhanced viral replication. Mechanistic analyses demonstrated that circRNA0857 functions as a "miRNA sponge," specifically targeting and sequestering miR1709 and miR1746. These miRNAs were found to regulate the expression of ATG3 and ATG7, respectively, two key autophagy-related genes in the autophagy pathway. By sponging miR1709 and miR1746, circRNA0857 upregulates the expression of ATG3 and ATG7, thereby enhancing autophagy and promoting NDV infection. This study is the first to identify circRNA0857 as a critical regulator in NDV infection, elucidating its role in enhancing NDV-induced autophagy via the miR1709/ATG3 and miR1746/ATG7 signaling axes. These findings significantly advance our understanding of how circRNAs regulate viral infections and provide novel insights into the molecular mechanisms underlying NDV replication.IMPORTANCECircular RNAs (circRNAs), as endogenous non-coding RNAs, are widely involved in various biological processes, particularly in the regulatory modulation of miRNA functions. Newcastle disease virus (NDV) can replicate in a variety of cells and promote its proliferation by hijacking and utilizing autophagy. In this study, we screened for novel circular RNAs that regulate NDV replication through modulating host autophagy and identified circRNA0857 as a key regulator of NDV infection. It enhances autophagy and promotes viral replication by acting as a "miRNA sponge," simultaneously targeting the two signaling axes miR1709/ATG3 and miR1746/ATG7. This research fills a gap in understanding how circRNAs regulate NDV-induced autophagy and provides a new perspective for understanding the interactions between host non-coding RNAs and viruses. This finding deepens our understanding of the pathogenic mechanisms of NDV and offers potential molecular targets for the development of antiviral strategies based on circular RNAs or miRNAs.

Anoikis is a specialized form of programmed cell death triggered by the detachment of cells from the extracellular matrix (ECM). Tumor cells that develop resistance to anoikis acquire the ability to detach, migrate, and colonize distant sites, ultimately leading to the formation of metastatic tumors. Bit1 (Bcl-2 inhibitor of transcription 1), a key effector of anoikis, is released into the cytoplasm upon loss of cell attachment and activates a caspase-independent pathway of apoptosis. Newcastle disease virus (NDV), a pathogen that poses a significant threat to the poultry industry, has also emerged as a promising oncolytic virus capable of selectively targeting and killing tumor cells. However, whether NDV can induce the death of anoikis-resistant tumor cells by activating Bit1 remains unclear. In this study, we utilized physical methods to induce cell suspension as a positive control for anoikis and further examined the expression and cellular localization of Bit1 following NDV infection in tumor cells. The results indicated that both viral infection and cell suspension resulted in partial cell death, accompanied by the translocation of Bit1 from the mitochondria to the cytoplasm and a reduction in its protein levels. Notably, Bit1 expression was found not to significantly affect viral replication. These findings suggest that NDV infection promotes tumor cell death by activating Bit1 translocation, mirroring the effects observed during cell suspension-induced anoikis. In addition, in vivo experiments demonstrated that NDV effectively inhibits the metastasis and growth of melanoma in mice, and that overexpression of Bit1 in tumor cells accelerates this process. This study provides novel insights into NDV-induced tumor cell death and identifies potential targets for understanding the mechanisms of oncolytic virus action.

Currently there are extremely few detailed reports on the quantitative detection of egg yolk immunoglobulin (IgY). Moreover, there is still a gap in standardized methods for IgY detection in poultry eggs. Size exclusion (SE) high performance liquid chromatography (HPLC) is a separation technique based on molecular size difference, and can be used as a standard method. Therefore, this study established a simple quantitative analysis method for IgY in various poultry eggs, based on the water dilution IgY extraction pretreatment, combined with SE-HPLC. The method was optimized for pretreatment and SE-HPLC detection conditions for five poultry eggs, and the analytical performance was evaluated by baseline separation. The results showed that poultry egg yolks diluted 10-fold with water and further purified by hexane could be used for SE-HPLC analysis. The optimized detection conditions for SE-HPLC were AdvanceBio SEC 300 A for the column, phosphate buffer containing 0.1 mol/L Na2SO4 (80 % mobile phase A) and acetonitrile (20 % mobile phase B) for the mobile phase at a flow rate of 0.8 mL/min, and a detection wavelength of 280 nm. The HPLC method showed good linearity (R-2 > 99 %), RSD < 10 %, satisfactory spiked recoveries (75.0 % similar to 105.3 %) and good reproducibility.

Infectious Bursal Disease (IBD) is an acute, highly contagious disease caused by IBDV, characterized by inflammation, atrophy of the Bursa of Fabricius, and immunosuppression. This study infected 21-day-old SPF chickens with three IBDV strains (classical YZ, very virulent AH, and variant SD). On day 7 post-infection, bursa samples were collected for transcriptomic and metabolomic analyses. Metabolite profiles were analyzed using multivariate statistics, and KEGG enrichment analysis was used to identify dysregulated pathways, elucidating the transcriptional and metabolic responses in IBDV-infected bursa tissue. Transcriptomic analysis identified 1733 DEGs in the YZ group, 5731 in the AH group, and 84 in the SD group. Gene Ontology clustering of common SDE genes between virus-infected and control groups focused on cellular components, molecular functions, and biological processes；KEGG enrichment showed they were mainly involved in lipid-related metabolic pathways for the three IBDV subtypes. Metabolomic analysis detected 460 significantly changed metabolites per subtype after IBDV infection. Lipid metabolism disorders were associated with IBDV, involving L-carnitine and other substances；KEGG analysis indicated the main pathways were lipid-related, like arachidonic acid (AA) metabolism. Moreover, this study verified mRNA levels of cytokines, NLRP3 protein level, and AA content. IBDV infection induces differential gene expression related to host immune response and metabolic regulation at the transcriptomic level, with metabolomic changes mainly involving lipid metabolism. Integrating transcriptomics and metabolomics provides a comprehensive understanding of the host's response to IBDV infection.

Red blood cells (RBCs) are the most abundant cell type in the blood and play a critical role as the primary carriers of oxygen to tissues and organs through blood circulation. The hemagglutinin-neuraminidase (HN) protein on the surface of the Newcastle disease virus (NDV) contains receptors that bind to the surface of RBCs, endowing NDV with agglutination properties that hold significant clinical diagnostic value. This raises an important question: could NDV bind to RBCs and use their carrier properties to facilitate transport to various tissues and organs? This study conducted both in vivo and in vitro experiments to confirm the adhesion and transport capabilities of chicken RBCs for the Newcastle disease virus. In addition, we found that NDV infection induces apoptosis in RBCs. These findings systematically explored the infection process of NDV in chicken RBCs and its subsequent effects, providing direct evidence of the potential role of chicken RBCs as a transport vehicle for the virus. This research offers a novel perspective on the mechanisms of NDV transmission.

Duck hepatitis A virus (DHAV) causes an acute and severe infectious disease characterized by liver swelling and hemorrhage, predominantly affecting ducklings under three weeks of age. This disease leads to significant economic losses in the duck farming industry. In China, both DHAV1 and DHAV3 are prevalent, with DHAV3 being more dominant. Among the three structural proteins of DHAV, the VP1 protein is the most critical as it induces neutralizing antibody production, serves as the binding protein for viral adsorption to cell-specific receptors, and determines viral antigenicity. The serum neutralization (SN) test is the ''gold standard'' for evaluating DHAV vaccine-immune serum; however, it is time-consuming and labor-intensive. To address this limitation, we developed a rapid, sensitive, and specific blocking ELISA (bELISA) for detecting DHAV3 antibodies. This assay utilizes DHAV3 virus-like particles (VLPs) as the coating antigen and the VP1-specific monoclonal antibody 4B8 as the blocking antibody. The bELISA demonstrated high sensitivity and specificity, detecting only DHAV3 antibodies without cross-reactivity with DHAV1 or other viral antibodies. The assay's cutoff value was determined to be 38.21 %, with intra- and inter-batch coefficients of variation below 5 %, indicating excellent reproducibility. The bELISA showed a 100 % positive concordance rate and a 93.65 % negative concordance rate with the SN test, resulting in an overall concordance rate of 96 %. In summary, this study presents the development of a high-quality bELISA for the detection of DHAV3 antibodies. This assay is suitable for clinical diagnosis of DHAV3, evaluation of maternal antibody levels, and assessment of vaccine efficacy in ducklings, offering a valuable tool for disease control and prevention in the duck industry.

Mitochondria and their electron transport chain (ETC) constitute the central machinery for cellular energy metabolism and biosynthetic regulation. Disruption of the ETC leads to reactive oxygen species (ROS) production and metabolic imbalance, but its precise role in viral replication and infection remains to be elucidated. In this study, we used Newcastle disease virus (NDV), an important avian pathogen and a promising oncolytic virus, as a model to explore its relationship with cellular mitochondrial metabolism. We demonstrate that NDV infection induces varying degrees of mitochondrial fragmentation, membrane potential dissipation, and ROS production, especially in p53-null H1299 cells compared to p53-wild-type A549 cells. ETC impairment restricts NDV replication primarily by limiting aspartate and pyrimidine nucleotide biosynthesis, rather than through ROS-mediated cytotoxicity or energy depletion. Notably, NDV replication in p53-null cells is highly sensitive to ETC complexes I and III inhibition, which can be rescued by exogenous aspartate or uridine supplementation. Mechanistically, p53 serves as a metabolic buffer, protecting mitochondrial function and maintaining precursor availability during viral infection. These findings elucidate the selective and differential utilization of mitochondrial ETC components by NDV and reveal that p53 status shapes cellular susceptibility to NDV-induced metabolic stress. Our work highlights mitochondrial metabolism and p53 as potential targets for antiviral and oncolytic strategies against NDV.IMPORTANCEThis study uncovers the intricate relationship between Newcastle disease virus (NDV) infection and host cell mitochondrial metabolism, with a particular emphasis on the pivotal regulatory role of p53. As both an important avian pathogen and a promising oncolytic virus, NDV disrupts mitochondrial function and the electron transport chain, leading to p53-mediated alterations in cellular energy metabolism and redox homeostasis. Our findings not only deepen the understanding of NDV-mitochondria interactions but also highlight the central role of p53 in viral infection and oncolytic mechanisms. These insights provide a theoretical foundation and novel therapeutic targets for antiviral and anticancer strategies based on p53 or mitochondrial pathways.

The endoribonuclease (EndoU) nsp15 of coronaviruses plays a crucial role in evading host innate immune responses by reducing the abundance of viral double-stranded RNA (dsRNA). However, our understanding of its interactions with host cellular targets remains limited. In this study, we demonstrate that overexpression of nsp15 from four coronavirus genera inhibits cellular protein synthesis and causes nuclear retention of PABPC1. Mutation analysis confirms the essential role of EndoU activity in these processes. Fluorescence in situ hybridization (FISH) analysis shows that cellular mRNA co-localizes with nsp15 in certain cells. Real time RT-PCR indicates that the mRNA levels of several antiviral genes decrease in cells expressing nsp15, and this reduction depends on the EndoU activity of nsp15. Using infectious bronchitis virus (IBV) as a model, we investigate the inhibitory effect of nsp15 on protein translation during infection. We find that infection with IBV with functional nsp15 suppresses protein synthesis in a PKR-eIF2α independent manner, with PABPC1 mainly located in the cytoplasm. However, infection with EndoU activity-deficiency mutant virus rIBV-nsp15-H238A results in the accumulation of viral dsRNA, triggering a PKR-eIF2α-dependent shutdown of protein synthesis and leading to the nuclear relocation of PABPC1. In the absence of the PKR-eIF2α pathway, IBV is still able to suppress host protein synthesis, while the inhibitory effect of rIBV-nsp15-H238A on protein synthesis was significantly reduced. Although nsp15 locates to replication-transcription complex (RTC) during infection, RNA immunoprecipitation (RIP)-Seq analysis confirms that IBV nsp15 binds to six viral RNAs and 237 cellular RNAs. The proteins encoded by the nsp15-associated cellular RNAs predominantly involved in translation. Additionally, proteomic analysis of the nsp15 interactome identifies 809 cellular proteins, which are significantly enriched in pathways related to ribosome biogenesis, RNA processing, and translation. Therefore, nsp15 helps virus circumvent the detrimental PKR-eIF2α pathway by reducing viral dsRNA accumulation and suppresses host protein synthesis by targeting host RNAs and proteins. This study reveals unique yet conserved mechanisms of protein synthesis shutdown by catalytically active nsp15 EndoU, shedding light on how coronaviruses regulate host protein expression.