Heterogeneous Nuclear Protein U Degraded the m6A Methylated TRAF3 Transcript by YTHDF2 To Promote Porcine Epidemic Diarrhea Virus Replication.

Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the Coronaviridae family and can cause fatal watery diarrhea in piglets, causing significant economic losses. Heterogeneous nuclear protein U (HNRNPU) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. However, it remains elusive whether and how cytoplasmic PEDV can be sensed by the RNA sensor HNRNPU. In this study we determined that HNRNPU was the binding partner of Nsp13 by immunoprecipitation-liquid chromatography-tandem mass spectrometry (IP/LC-MS/MS) analysis. The interaction between Nsp13 and HNRNPU was demonstrated by using coimmunoprecipitation and confocal immunofluorescence. Next, we identified that HNRNPU expression is significantly increased during PEDV infection, whereas the transcription factor hepatocyte nuclear factor 1α (HNF1A) could negatively regulate HNRNPU expression. HNRNPU was retained in the cytoplasm by interaction with PEDV Nsp13. We found that HNRNPU overexpression effectively facilitated PEDV replication, while knockdown of HNRNPU impaired viral replication, suggesting a promoting function of HNRNPU to PEDV infection. Additionally, HNRNPU was found to promote PEDV replication by affecting TRAF3 degradation at the transcriptional level to inhibit PEDV-induced beta interferon (IFN-ß) production. Mechanistically, HNRNPU downregulates TRAF3 mRNA levels via the METTL3-METTL14/YTHDF2 axis and regulates immune responses through YTHDF2-dependent mRNA decay. Together, our findings reveal that HNRNPU serves as a negative regulator of innate immunity by degrading TRAF3 mRNA in a YTHDF2-dependent manner and consequently facilitating PEDV propagation. Our findings provide new insights into the immune escape of PEDV. IMPORTANCE PEDV, a highly infectious enteric coronavirus, has spread rapidly worldwide and caused severe economic losses. During virus infection, the host regulates innate immunity to inhibit virus infection. However, PEDV has evolved a variety of different strategies to suppress host IFN-mediated antiviral responses. Here, we identified that HNRNPU interacted with viral protein Nsp13. HNRNPU protein expression was upregulated, and the transcription factor HNF1A could negatively regulate HNRNPU expression during PEDV infection. HNRNPU also downregulated TRAF3 mRNA through the METTL3-METTL14/YTHDF2 axis to inhibit the production of IFN-ß and downstream antiviral genes in PEDV-infected cells, thereby promoting viral replication. Our findings reveal a new mechanism with which PEDV suppresses the host antiviral response.

Passive localization and reconstruction of multiple non-line-of-sight objects in a scene with a large visible transmissive window.

Passive non-line-of-sight imaging methods have been demonstrated to be capable of reconstructing images of hidden objects. However, current passive non-line-of-sight imaging methods have performance limitations due to the requirements of an occluder and aliasing between multiple objects. In this paper, we propose a method for passive localization and reconstruction of multiple non-line-of-sight objects in a scene with a large visible transmissive window. The analysis of the transport matrix revealed that more redundant information is acquired in a scene with a window than that with an occluder, which makes the image reconstruction more difficult. We utilized the projection operator and residual theory to separate the reconstruction equation of multiple objects into the independent equations of the located objects that can be reconstructed independently by TVAL3 and Split-Bregman algorithms, which greatly reduces the computational complexity of the reconstruction. Our method lays the foundation for multiple objects reconstruction in complex non-line-of-sight scenes.

High-resolution non-confocal non-line-of-sight imaging based on spherical-slice transform from spatial and temporal frequency to space and time.

Current non-confocal non-line-of-sight (NLOS) imaging faces the problems of low resolution and limited scene adaptability. We propose a non-confocal NLOS imaging method based on spherical-slice transform from spatial and temporal frequency to space and time. Simulation and experimental results show that the proposed method has high-resolution reconstruction without artifact interference, shape distortion, and position offset. Furthermore, it has strong scene adaptability. After GPU acceleration, the reconstruction time of the proposed method can be reduced to several hundred milliseconds for the PF32 photon array camera with 32 × 32 detection units. In the future, the proposed method has great potential for application in real-time NLOS imaging systems.

Biogated mesoporous silica nanoagents for inhibition of cell migration and combined cancer therapy.

Migration is an initial step in tumor expansion and metastasis; suppressing cellular migration is beneficial to cancer therapy. Herein, we designed a novel biogated nanoagents that integrated the migration inhibitory factor into the mesoporous silica nanoparticle (MSN) drug delivery nanosystem to realize cell migratory inhibition and synergistic treatment. Antisense oligonucleotides (Anti) of microRNA-330-3p, which is positively related with cancer cell proliferation, migration, invasion, and angiogenesis, not only acted as the locker for blocking drugs but also acted as the inhibitory factor for suppressing migration via gene therapy. Synergistic with gene therapy, the biogated nanoagents (termed as MSNs-Gef-Anti) could achieve on-demand drug release based on the intracellular stimulus-recognition and effectively kill tumor cells. Experimental results synchronously demonstrated that the migration suppression ability of MSNs-Gef-Anti nanoagents (nearly 30%) significantly contributed to cancer therapy, and the lethality rate of the non-small-cell lung cancer was up to 70%. This strategy opens avenues for realizing efficacious cancer therapy and should provide an innovative way for pursuing the rational design of advanced nano-therapeutic platforms with the combination of cancer cell migratory inhibition.

The novel Nsp9-interacting host factor H2BE promotes PEDV replication by inhibiting endoplasmic reticulum stress-mediated apoptosis.

Porcine epidemic diarrhoea (PED) caused by porcine epidemic diarrhoea virus (PEDV) has led to significant economic losses in the swine industry worldwide. Histone Cluster 2, H2BE (HIST2H2BE), the main protein component in chromatin, has been proposed to play a key role in apoptosis. However, the relationship between H2BE and PEDV remains unclear. In this study, H2BE was shown to bind and interact with PEDV nonstructural protein 9 (Nsp9) via immunoprecipitation-mass spectrometry (IP-MS). Next, we verified the interaction of Nsp9 with H2BE by immunoprecipitation and immunofluorescence. H2BE colocalized with Nsp9 in the cytoplasm and nuclei. PEDV Nsp9 upregulated the expression of H2BE by inhibiting the expression of IRX1. We demonstrated that overexpression of H2BE significantly promoted PEDV replication, whereas knockdown of H2BE by small interfering RNA (siRNA) inhibited PEDV replication. Overexpression of H2BE led to significantly inhibited GRP78 expression, phosphorylated PERK (p-PERK), phosphorylated eIF2 (p-eIF2), phosphorylated IRE1 (p-IRE1), and phosphorylated JNK (p-JNK); negatively regulated CHOP and Bax expression and caspase-9 and caspase-3 cleavage; and promoted Bcl-2 production. Knocking down H2BE exerted the opposite effects. Furthermore, we found that after deletion of amino acids 1-28, H2BE did not promote PEDV replication. In conclusion, these studies revealed the mechanism by which H2BE is associated with ER stress-mediated apoptosis to regulate PEDV replication. Nsp9 upregulates H2BE. H2BE plays a role in inhibiting apoptosis and thus facilitating viral replication, which depends on the N-terminal region of H2BE (amino acids 1-28). These findings provide a reference for host-PEDV interactions and offer the possibility for developing strategies for PEDV decontamination and prevention.

Differential expression analysis of mRNAs, lncRNAs, and miRNAs expression profiles and construction of ceRNA networks in PEDV infection.

BACKGROUND: Porcine Epidemic Diarrhea Virus (PEDV) is a coronavirus that seriously affects the swine industry. MicroRNAs and long noncoding RNAs are two relevant non-coding RNAs (ncRNAs) class and play crucial roles in a variety of physiological processes. Increased evidence indicates a complex interaction between mRNA and ncRNA. However, our understanding of the function of ncRNA involved in host-PEDV interaction is limited. RESULTS: A total of 1,197 mRNA transcripts, 539 lncRNA transcripts, and 208 miRNA transcripts were differentially regulated at 24 h and 48 h post-infection. Gene ontology (GO) and KEGG pathway enrichment analysis showed that DE mRNAs and DE lncRNAs were mainly involved in biosynthesis, innate immunity, and lipid metabolism. Moreover, we constructed a miRNA-mRNA-pathway network using bioinformatics, including 12 DE mRNAs, 120 DE miRNAs, and 11 pathways. Finally, the target genes of DE miRNAs were screened by bioinformatics, and we constructed immune-related lncRNA-miRNA-mRNA ceRNA networks. Then, the selected DE genes were validated by qRT-PCR, which were consistent with the results from RNA-Seq data. CONCLUSIONS: This study provides the comprehensive analysis of the expression profiles of mRNAs, lncRNAs, and miRNAs during PEDV infection. We characterize the ceRNA networks which can provide new insights into the pathogenesis of PEDV.

TRIM56 overexpression restricts porcine epidemic diarrhoea virus replication in Marc-145 cells by enhancing TLR3-TRAF3-mediated IFN-ß antiviral response.

Infection with the porcine epidemic diarrhoea virus (PEDV) causes severe enteric disease in suckling piglets, causing massive economic losses in the swine industry worldwide. Tripartite motif-containing 56 (TRIM56) has been shown to augment type I IFN response, but whether it affects PEDV replication remains uncharacterized. Here we investigated the role of TRIM56 in Marc-145 cells during PEDV infection. We found that TRIM56 expression was upregulated in cells infected with PEDV. Overexpression of TRIM56 effectively reduced PEDV replication, while knockdown of TRIM56 resulted in increased viral replication. TRIM56 overexpression significantly increased the phosphorylation of IRF3 and NF-κB P65, and enhanced the IFN-ß antiviral response, while silencing TRIM56 did not affect IRF3 activation. TRIM56 overexpression increased the protein level of TRAF3, the component of the TLR3 pathway, thereby significantly activating downstream IRF3 and NF-κB signalling. We demonstrated that TRIM56 overexpression inhibited PEDV replication and upregulated expression of IFN-ß, IFN-stimulated genes (ISGs) and chemokines in a dose-dependent manner. Moreover, truncations of the RING domain, N-terminal domain or C-terminal portion on TRIM56 were unable to induce IFN-ß expression and failed to restrict PEDV replication. Together, our results suggested that TRIM56 was upregulated in Marc-145 cells in response to PEDV infection. Overexpression of TRIM56 inhibited PEDV replication by positively regulating the TLR3-mediated antiviral signalling pathway. These findings provide evidence that TRIM56 plays a positive role in the innate immune response during PEDV infection.

Metabolic indexes of obesity in patients with common mental disorders in stable stage.

BACKGROUND: Obesity is a serious worldwide public health problem, especially for people with mental disorders. AIM: To explore the related factors of obesity by analyzing the metabolic indexes of patients with common mental disorders in stable stage. METHODS: Five hundred seventy-six subjects with major depressive disorder (MDD), bipolar disorder (BD) or schizophrenia (SCZ) were included, who received fixed drug dose and routine drug treatment for 2 years or more. Their venous blood was collected, and the blood metabolic indexes were analyzed. RESULTS: BD and SCZ are more prone to obesity than MDD. Multiple linear regression analysis showed that the value of BMI increased with the increase of age(B = 0.084, p < 0.001), TG(B = 0.355, p = 0.024), LDL(B = 0.697, p < 0.001), LDH(B = 0.011, p = 0.002), SCr(B = 0.051, p < 0.001), UA(B = 0.014, p < 0.001), HbA1c(B = 0.702, p = 0.004) and hsCRP(B = 0.101, p < 0.001). And It decreased with the increase of HDL(B = -1.493, p < 0.001). DISCUSSION: People with mental disorders should regularly check blood indicators and strengthen weight management to reduce the risk of obesity and promote their health.

Somatic MIWI2 Hinders Direct Lineage Reprogramming From Fibroblast to Hepatocyte.

Remodeling of the gene regulatory network in cells is believed to be a prerequisite for their lineage reprogramming. However, its key regulatory factors are not yet elucidated. In this article, we investigate the role of PIWI proteins and provide evidence that one of them, MIWI2, is elicited during transdifferentiation of fibroblasts into hepatocyte-like cells. In coincidence with the peak expression of MIWI2, we identified the appearance of a unique intermediate epigenetic state characterized by a specific Piwi-interacting RNA (piRNA) profile consisting of 219 novel sequences. Knockout of MIWI2 greatly improved the formation of the induced hepatocytes, whereas overexpression of exogenous MIWI2 completely abolished the stimulated effect. A bioinformatics analysis of piRNA interaction network, followed by experimental validation, revealed the Notch signaling pathway as one of the immediate effectors of MIWI2. Altogether, our results show for the first time that temporal expression of MIWI2 contributes negatively to cell plasticity not only in germline, but also in developed cells, such as mouse fibroblasts. Stem Cells 2019;37:803-812.

Characterization and Complete Genome Analysis of Pseudomonas aeruginosa Bacteriophage vB_PaeP_LP14 Belonging to Genus Litunavirus.

A lytic Pseudomonas aeruginosa phage vB_PaeP_LP14 belonging to the family Podoviridae was isolated from infected mink. The microbiological characterization revealed that LP14 was stable at 40 to 50 °C and stable over a broad range of pH (5 to 12). The latent period was 5 min, and the burst size was 785 pfu/infected cell. The whole-genome sequencing showed that LP14 was a dsDNA virus and has a genome of 73,080 bp. The genome contained 93 predicted open reading frames (ORFs), 17 of which have known functions including DNA replication and modification, transcriptional regulation, structural and packaging proteins, and host cell lysis. No tRNA genes were identified. BLASTn analysis revealed that phage LP14 had a high-sequence identity (96%) with P. aeruginosa phage YH6. Both morphological characterization and genome annotation indicate that phage LP14 is a memberof the family Podoviridae genus Litunavirus. The study of phage LP14 will provide basic information for further research on treatment of P. aeruginosa infections.

State-of-the-Art Internet of Things in Protected Agriculture.

The Internet of Things (IoT) has tremendous success in health care, smart city, industrial production and so on. Protected agriculture is one of the fields which has broad application prospects of IoT. Protected agriculture is a mode of highly efficient development of modern agriculture that uses artificial techniques to change climatic factors such as temperature, to create environmental conditions suitable for the growth of animals and plants. This review aims to gain insight into the state-of-the-art of IoT applications in protected agriculture and to identify the system structure and key technologies. Therefore, we completed a systematic literature review of IoT research and deployments in protected agriculture over the past 10 years and evaluated the contributions made by different academicians and organizations. Selected references were clustered into three application domains corresponding to plant management, animal farming and food/agricultural product supply traceability. Furthermore, we discussed the challenges along with future research prospects, to help new researchers of this domain understand the current research progress of IoT in protected agriculture and to propose more novel and innovative ideas in the future.

Platelet Surface CD62p and Serum Vitamin D Levels are Associated with Clopidogrel Resistance in Chinese Patients with Ischemic Stroke.

BACKGROUND: To explore the association of platelet activation markers, vitamin D, and antiplatelet drugs resistance in ischemic stroke patients. METHODS: A total of 230 patients with ischemic stroke were enrolled in this study. Platelet aggregation, platelet activation marker (CD62p), and vitamin D were measured after 7-14 days of dual antiplatelet treatment (aspirin + clopidogrel). All individuals were divided into a drug resistance group and a drug sensitive group according to the platelet maximum aggregation rate induced by antagonist adenosine diphosphate or arachidonic acid. RESULTS: In this study, the prevalence of aspirin resistance was low (1.2%), while the prevalence of clopidogrel resistance (CR) was 24.8%, so we focused on CR. The percentage of CD62p on activated platelet [(25.74 ± 4.61) versus (12.41 ± 3.93), P < .001] and the prevalence of hypertension [93.0% (53) versus 79.8% (138), P = .021] in CR group were significantly higher than those in clopidogrel sensitive (CS) group, while the vitamin D concentration [(8.96 ± 4.41) versus (13.9 ± 4.84) ng/mL, P = .003] in CR group was significantly lower compared with the CS group. No significant difference was found in soluble P-selectin between these 2 groups [(56.2 ± 16.13) versus (54.2 ± 14.87) ng/mL, P = .258], neither in calcium [(2.29 ± .12) versus (2.33 ± .13) mmol/L, P = .821]. Logistic regression analysis showed that hypertension (odds ratio [OR] = 5.348, 95% confidence intervals [CI] 1.184-23.350, P = .026), expression of platelet CD62p (OR = 1.095, 95% CI 1.052-1.201, P = .018) and vitamin D level (OR = .832, 95% CI .763-.934, P = .005) were associated with CR in ischemic stroke patients. CONCLUSIONS: CR in ischemic stroke patients is associated with several independent predictors, including increased platelet activation marker CD62p, decreased vitamin D level, and hypertension.

High-Pressure Homogenization Pre-Treatment Improved Functional Properties of Oyster Protein Isolate Hydrolysates.

The effects of HPH (high-pressure homogenization) pre-treatment on the functional properties of OPIH (oyster protein isolates hydrolysates) were studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles, solubility, particle size distribution, zeta potential, surface hydrophobicity, emulsifying activity index and microstructure of emulsions were analyzed. Results indicated that HPH pre-treatment increased the accessibility of OPI to trypsin hydrolysis, resulting in decease in particle size, increase in solubility, absolute zeta potential, surface hydrophobicity and emulsifying activity index. In addition, HPH pre-treated OPIH emulsions became more uniform and the particle size of droplets decreased. These results revealed that HPH pre-treatment has the potential to modify the functional properties of OPIH.

BCL6 mediates the effects of Gastrodin on promoting M2-like macrophage polarization and protecting against oxidative stress-induced apoptosis and cell death in macrophages.

Cerebral palsy (CP) is the most common childhood disability worldwide, yet biomarkers for predicting CP are lacking. By subjecting peripheral blood samples from 62 CP patients and 30 healthy controls to Affymetrix GeneChip® PrimeView™ HumanGene Expression Microarray analysis, we identified the novel biomarker B-cell lymphoma 6 (BCL6) as the most upregulated gene in the CP samples. Gastrodin is a traditional Chinese medicine and bioactive compound that promotes adductor angle release, as well as gross and fine motor performance by increasing Gross Motor Function Measure-66 and Fine Motor Function Measure-45 scores. Gastrodin upregulates the mRNA expression of Mgl2 and Mrc1, M2 macrophage markers, and arginase activity, an M2 polarization indicator, in murine RAW264.7 macrophages. Moreover, these effects were blocked by BCL6 siRNA, which also abrogated the protective effects of Gastrodin against hydrogen peroxide-induced apoptosis and death in RAW264.7 cells. Our work identified BCL6 as a novel biomarker for early prediction of CP. Moreover, we demonstrated that Gastrodin not only stimulated polarization toward M2-like macrophages, which promote tissue repair, but also rescued macrophages from oxidative stress, apoptosis and death by inducing BCL6 expression. BCL6-targeted therapeutic strategies have promise for improving motor performance in CP patients.

Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes.

Selection of Leptin Surrogates by a General Phenotypic Screening Method for Receptor Agonists.

There is a high demand for agonist biomolecules such as cytokine surrogates in both biological and medicinal research fields. These are typically sourced through natural ligand engineering or affinity-based screening, followed by individual functional validation. However, efficient screening methods for identifying rare hits within immense libraries are very limited. In this research article, we introduce a phenotypic screening method utilizing biological receptor activation-dependent cell survival (BRADS). This method offers a high-throughput, low-background, and cost-effective approach that can be implemented in virtually any biochemical laboratory setting. As a proof-of-concept, we successfully identified a surrogate for human leptin following a two-week cell culture process, without the need for specialized high-throughput equipment or reagents. This surrogate effectively emulates the activity of native human leptin in cell validation assays. Our findings not only underscore the effectiveness of BRADS but also suggest its potential applicability to a broad range of biological receptors, including Notch and GPCRs.

PEDV inhibits HNRNPA3 expression by miR-218-5p to enhance cellular lipid accumulation and promote viral replication.

Porcine epidemic diarrhea virus (PEDV) requires complete dependence on the metabolic system of the host cell to complete its life cycle. There is a strong link between efficient viral replication and cellular lipid synthesis. However, the mechanism by which PEDV interacts with host cells to hijack cellular lipid metabolism to promote its replication remains unclear. In this study, PEDV infection significantly enhanced the expression of lipid synthesis-related genes and increased cellular lipid accumulation. Furthermore, using liquid chromatography-tandem mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) as the interacting molecule of PEDV NSP9. We demonstrated that the expression of HNRNPA3 was downregulated by PEDV-induced miR-218-5p through targeting its 3' untranslated region. Interestingly, knocking down HNRNPA3 facilitated the PEDV replication by promoting cellular lipid synthesis. We next found that the knockdown of HNRNPA3 potentiated the transcriptional activity of sterol regulatory element-binding transcription factor 1 (SREBF1) through zinc finger protein 135 (ZNF135) as well as PI3K/AKT and JNK signaling pathways. In summary, we propose a model in which PEDV downregulates HNRNPA3 expression to promote the expression and activation of SREBF1 and increase cellular lipid accumulation, providing a novel mechanism by which PEDV interacts with the host to utilize cellular lipid metabolism to promote its replication.IMPORTANCEAs the major components and structural basis of the viral replication complexes of positive-stranded RNA viruses, lipids play an essential role in viral replication. However, how PEDV manipulates host cell lipid metabolism to promote viral replication by interacting with cell proteins remains poorly understood. Here, we found that SREBF1 promotes cellular lipid synthesis, which is essential for PEDV replication. Moreover, HNRNPA3 negatively regulates SREBF1 activation and specifically reduces lipid accumulation, ultimately inhibiting PEDV dsRNA synthesis. Our study provides new insight into the mechanisms by which PEDV hijacks cell lipid metabolism to benefit viral replication, which can offer a potential target for therapeutics against PEDV infection.

Genome-wide screen reveals cellular functions that counteract rifampicin lethality in Escherichia coli.

IMPORTANCE: Rifamycins are a group of antibiotics with a wide antibacterial spectrum. Although the binding target of rifamycin has been well characterized, the mechanisms underlying the discrepant killing efficacy between gram-negative and gram-positive bacteria remain poorly understood. Using a high-throughput screen combined with targeted gene knockouts in the gram-negative model organism Escherichia coli, we established that rifampicin efficacy is strongly dependent on several cellular pathways, including iron acquisition, DNA repair, aerobic respiration, and carbon metabolism. In addition, we provide evidence that these pathways modulate rifampicin efficacy in a manner distinct from redox-related killing. Our findings provide insights into the mechanism of rifamycin efficacy and may aid in the development of new antimicrobial adjuvants.

Nsp10-interacting host protein SAP18 restricts PEDV replication in Marc-145 cells via enhancing dephosphorylation of RIG-I.

PEDV, a single-stranded RNA virus, causes significant economic losses in the pig industry. Sin3-associated protein 18 (SAP18) is known for its role in transcriptional inhibition and RNA splicing. However, research on SAP18's involvement in PEDV infection is limited. Here, we identified an interaction between SAP18 and PEDV nonstructural protein 10 (Nsp10) using immunoprecipitation-mass spectrometry (IP-MS) and confirmed it through immunoprecipitation and laser confocal microscopy. Additionally, PEDV Nsp10 reduced SAP18 protein levels and induced its cytoplasmic accumulation. Overexpressing SAP18 suppressed PEDV replication, meanwhile its knockdown via short interfering RNA (siRNA) enhanced replication. SAP18 overexpression boosted IRF3 and NF-κB P65 phosphorylation, nuclear translocation, and IFN-ß antiviral response. Furthermore, SAP18 upregulated RIG-I expression and facilitated its dephosphorylation, while SAP18 knockdown had the opposite effect. Finally, SAP18 interacted with phosphatase 1 (PP1) catalytic subunit alpha (PPP1CA), promoting PPP1CA-RIG-I interaction during PEDV infection. These findings highlight SAP18's role in activating the type I interferon pathway and inhibiting viral replication by promoting RIG-I dephosphorylation through its interaction with PPP1CA.

GRAMD4 regulates PEDV-induced cell apoptosis inhibiting virus replication via the endoplasmic reticulum stress pathway.

Porcine epidemic diarrhea (PED) caused by the porcine epidemic diarrhea virus (PEDV) has caused huge losses in the swine industry worldwide. Glucosyltransferase Rab-like GTPase activator and myotubularin domain containing 4 (GRAMD4) is a proapoptotic protein, which replaced p53 inducing mitochondrial apoptosis. However, the relationship between GRAMD4 and PEDV has not been reported. Here, we aimed to investigate the potential role of GRAMD4 during PEDV infection. In this study, we used co-immunoprecipitation (co-IP) and mass spectrometry to identify GRAMD4 interaction with PEDV non-structural protein 6 (NSP6). Immunoprecipitation and laser confocal microscopy were utilized to demonstrate that GRAMD4 interacts with NSP6. NSP6 reduces GRAMD4 production through PERK and IRE1 pathway-mediated apoptosis. We demonstrated that overexpression of GRAMD4 effectively impaired the replication of PEDV, whereas knockdown of GRAMD4 facilitated the replication of PEDV. Overexpression of GRAMD4 increased GRP78, phosphorylated PERK (p-PERK), phosphorylated IRE1(p-IRE1) levels, promoted CHOP, phosphorylated JNK (p-JNK), Bax expression, caspase 9 and caspase 3 cleavage, and inhibited Bcl-2 production. Knockdown of GRAMD4 has the opposite effect. Finally, deletion of the GRAM domain of GRAMD4 cannot cause endoplasmic reticulum stress (ER stress)-mediated apoptosis and inhibit virus replication. In conclusion, these studies revealed the mechanism by which GRAMD4 was associated with ER stress and apoptosis regulating PEDV replication. NSP6 acted as a potential down-regulator of GRAMD4 and promoted the degradation of GRAMD4. GRAMD4 played a role in facilitating apoptosis and restricting virus replication, and the GRAM domain was required. These findings provided a reference for host-PEDV interactions and offered the possibility for PEDV decontamination and prevention.