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Interactions between double-stranded RNA (dsRNA) and proteins play an important role in cellular homeostasis by regulating the editing, stability, and splicing of intracellular RNA. The identification of dsRNA-binding proteins (dsRBPs) is key; however, it has long been challenging to purify dsRBPs from cells. In this study, we developed a novel method, dsRBPC (dsRNA-binding protein capture), to purify cellular dsRBPs based on classic phase separation purification procedures. A global dsRNA-binding proteome of LLC-PK1 cells was obtained, and we identified 1326 dsRBPs, including 1303 putative novel dsRBPs. Functional analyses suggested that these enriched dsRBPs are mainly associated with rRNA processing, RNA splicing, transcriptional regulation, and nucleocytoplasmic transport. We also found that the ARM (armadillo/beta-catenin-like repeats) motif is a previously unknown dsRNA-binding domain, as demonstrated by biochemical experiments. Collectively, this study provides a useful approach for dsRBP identification and the discovery of a global dsRNA-binding proteome to comprehensively map the dsRNA - protein interaction network.
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ARN Bicatenario , Proteínas de Unión al ARN , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/química , Animales , Proteoma/metabolismo , Unión Proteica , Porcinos , Línea Celular , Empalme del ARN , Separación de FasesRESUMEN
Porcine reproductive and respiratory syndrome (PRRS), which poses substantial threats to the global pig industry, is primarily characterized by interstitial pneumonia. Cluster of differentiation 163 (CD163) is the essential receptor for PRRSV infection. Metalloproteinase-mediated cleavage of CD163 leads to the shedding of soluble CD163 (sCD163), thereby inhibiting PRRSV proliferation. However, the exact cleavage site in CD163 and the potential role of sCD163 in inflammatory responses during PRRSV infection remain unclear. Herein, we found that PRRSV infection increased sCD163 levels, as demonstrated in primary alveolar macrophages (PAMs), immortalized PAM (IPAM) cell lines, and sera from PRRSV-infected piglets. With LC-MS/MS, Arg-1041/Ser-1042 was identified as the cleavage site in porcine CD163, and an IPAM cell line with precise mutation at the cleavage site was constructed. Using the precisely mutated IPAM cells, we found that exogenous addition of sCD163 protein promoted inflammatory responses, while mutation at the CD163 cleavage site suppressed inflammatory responses. Consistently, inhibition of sCD163 using its neutralizing antibodies reduced PRRSV infection-triggered inflammatory responses. Importantly, sCD163 promoted cell polarization from M2 to M1 phenotype, which in turn facilitated inflammatory responses. Taken together, our findings identify sCD163 as a novel proinflammatory mediator and provide valuable insights into the mechanisms underlying the induction of inflammatory responses by PRRSV infection.
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Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Inflamación , Macrófagos Alveolares , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Receptores de Superficie Celular , Animales , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Porcinos , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Macrófagos Alveolares/virología , Macrófagos Alveolares/inmunología , Inflamación/virología , Línea CelularRESUMEN
Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. The coronavirus spike (S) glycoprotein, especially the S1 subunit, mediates viral entry by binding to cellular receptors. However, the functional receptor of PDCoV remains poorly understood. In this study, we used the soluble PDCoV S1 protein as bait to capture the S1-binding cellular transmembrane proteins in combined immunoprecipitation and mass spectrometry analyses. A single guide RNA screen identified d-glucuronyl C5-epimerase (GLCE), a heparan sulfate-modifying enzyme, as a proviral host factor for PDCoV infection. GLCE knockout significantly inhibited the attachment and internalization stages of PDCoV infection. We also demonstrated the interaction between GLCE and PDCoV S with coimmunoprecipitation in both an overexpression system and PDCoV-infected cells. GLCE could be localized to the cell membrane, and an anti-GLCE antibody suppressed PDCoV infection. Although GLCE expression alone did not render nonpermissive cells susceptible to PDCoV infection, GLCE promoted the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. In conclusion, our results demonstrate that GLCE is a novel cell-surface factor facilitating PDCoV entry and provide new insights into PDCoV infection. IMPORTANCE: The identification of viral receptors is of great significance, potentially extending our understanding of viral infection and pathogenesis. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus with the potential for cross-species transmission. However, the receptors or coreceptors of PDCoV are still poorly understood. The present study confirms that d-glucuronyl C5-epimerase (GLCE) is a positive regulator of PDCoV infection, promoting viral attachment and internalization. The anti-GLCE antibody suppressed PDCoV infection. Mechanically, GLCE interacts with PDCoV S and promotes the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. This work identifies GLCE as a novel cell-surface factor facilitating PDCoV entry and paves the way for further insights into the mechanisms of PDCoV infection.
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Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant threat to the global swine industry. The development of highly effective subunit nanovaccines is a promising strategy for preventing PRRSV variant infections. In this study, two different types of ferritin (Ft) nanovaccines targeting the major glycoprotein GP5, named GP5m-Ft and (Bp-IVp)3-Ft, were constructed and evaluated as vaccine candidates for PRRSV. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) demonstrated that both purified GP5m-Ft and (Bp-IVp)3-Ft proteins could self-assemble into nanospheres. A comparison of the immunogenicity of GP5m-Ft and (Bp-IVp)3-Ft with an inactivated PRRSV vaccine in BALB/c mice revealed that mice immunized with GP5m-Ft exhibited the highest ELISA antibody levels, neutralizing antibody titers, the lymphocyte proliferation index, and IFN-γ levels. Furthermore, vaccination with the GP5m-Ft nanoparticle effectively protected piglets against a highly pathogenic PRRSV challenge. These findings suggest that GP5m-Ft is a promising vaccine candidate for controlling PRRS.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Ferritinas , Ratones Endogámicos BALB C , Nanopartículas , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Proteínas del Envoltorio Viral , Vacunas Virales , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Ferritinas/inmunología , Porcinos , Ratones , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Nanopartículas/química , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Femenino , Interferón gamma/metabolismo , NanovacunasRESUMEN
Since it was first reported in 2013, the NADC30-like PRRSV has been epidemic in China. Hubei Province is known as China's key hog-exporting region. To understand the prevalence and genetic variation of PRRSV, herein, we detected and analyzed 317 lung tissue samples from pigs with respiratory disease in Hubei Province, and demonstrated that the NADC30-like strain was the second-most predominant strain during 2017-2018, following the highly pathogenic PRRSV (HP-PRRSV). Additionally, we isolated a new NADC30-like PRRSV strain, named CHN-HB-2018, which could be stably passaged in Marc-145 cells. Genetic characterization analysis showed that compared with the NADC30 strain, the CHN-HB-2018 strain had several amino acid variations in glycoprotein (GP) 3, GP5, and nonstructural protein 2 (NSP2). Moreover, the CHN-HB-2018 strain showed a unique 5-amino acid (aa) deletion in NSP2, which has not previously been reported. Gene recombination analysis identified the CHN-HB-2018 strain as a potentially recombinant PRRSV of the NADC30-like strain and HP-PRRSV. Animal experiments indicated that the CHN-HB-2018 strain has a mild pathogenicity, with no mortality and only mild fever observed in piglets. This study contributes to defining the evolutionary characteristics of PRRSV and its molecular epidemiology in Hubei Province, and provides a potential candidate strain for PRRSV vaccine development.
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Filogenia , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Virus del Síndrome Respiratorio y Reproductivo Porcino/clasificación , Animales , Porcinos , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/epidemiología , China/epidemiología , Virulencia , Genoma Viral , Recombinación Genética , Variación Genética , Pulmón/virología , Pulmón/patologíaRESUMEN
Orf virus (ORFV), a member of the genus Parapoxvirus, possesses an excellent immune activation capability, which makes it a promising immunomodulation agent. In this study, we evaluated ORFV as a novel adjuvant to enhance the immune response of mice to a subunit vaccine using porcine circovirus type 2 (PCV2) capsid (Cap) protein as a model. Our results showed that both inactivated and live attenuated ORFV activated mouse bone marrow-derived dendritic cells and increased expression of immune-related cytokines interleukin (IL)-1ß, IL-6, and TNF-α. Enhanced humoral and cellular immune responses were induced in mice immunized with PCV2 Cap protein combined with inactivated or live attenuated ORFV adjuvant compared with the aluminum adjuvant. Increased secretion of Th1 and Th2 cytokines by splenic lymphocytes in immunized mice further indicated that the ORFV adjuvant promoted a mixed Th1/Th2 immune response. Moreover, addition of the ORFV adjuvant to the PCV2 subunit vaccine significantly reduced the viral load in the spleen and lungs of PCV2-challenged mice and prevented pathological changes in lungs. This study demonstrates that ORFV enhances the immunogenicity of a PCV2 subunit vaccine by improving the adaptive immune response, suggesting the potential application of ORFV as a novel adjuvant.
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Adyuvantes Inmunológicos , Infecciones por Circoviridae , Circovirus , Citocinas , Virus del Orf , Vacunas de Subunidad , Vacunas Virales , Animales , Circovirus/inmunología , Ratones , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Infecciones por Circoviridae/prevención & control , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/inmunología , Infecciones por Circoviridae/virología , Adyuvantes Inmunológicos/administración & dosificación , Citocinas/inmunología , Virus del Orf/inmunología , Proteínas de la Cápside/inmunología , Femenino , Inmunidad Celular , Células Dendríticas/inmunología , Carga Viral , Anticuerpos Antivirales/sangre , Inmunidad Humoral , Porcinos , Adyuvantes de Vacunas , Ratones Endogámicos BALB C , Células TH1/inmunologíaRESUMEN
Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.
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Proteasas 3C de Coronavirus , Virus de la Diarrea Epidémica Porcina , Animales , Humanos , Proteasas 3C de Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/veterinaria , Células HEK293 , Interferón Tipo I/metabolismo , Proteolisis , Proteoma/metabolismo , Especificidad por Sustrato , Porcinos , Proteínas Virales/metabolismo , Proteínas Virales/genética , Replicación ViralRESUMEN
BACKGROUND: Migraine stands as a prevalent primary headache disorder, with prior research highlighting the significant involvement of oxidative stress and inflammatory pathways in its pathogenesis and chronicity. Existing evidence indicates the capacity of Dl-3-n-butylphthalide (NBP) to mitigate oxidative stress and inflammation, thereby conferring neuroprotective benefits in many central nervous system diseases. However, the specific therapeutic implications of NBP in the context of migraine remain to be elucidated. METHODS: We established a C57BL/6 mouse model of chronic migraine (CM) using recurrent intraperitoneal injections of nitroglycerin (NTG, 10 mg/kg), and prophylactic treatment was simulated by administering NBP (30 mg/kg, 60 mg/kg, 120 mg/kg) by gavage prior to each NTG injection. Mechanical threshold was assessed using von Frey fibers, and photophobia and anxious behaviours were assessed using a light/dark box and elevated plus maze. Expression of c-Fos, calcitonin gene-related peptide (CGRP), Nucleus factor erythroid 2-related factor 2 (Nrf2) and related pathway proteins in the spinal trigeminal nucleus caudalis (SP5C) were detected by Western blotting (WB) or immunofluorescence (IF). The expression of IL-1ß, IL-6, TNF-α, Superoxide dismutase (SOD) and malondialdehyde (MDA) in SP5C and CGRP in plasma were detected by ELISA. A reactive oxygen species (ROS) probe was used to detect the expression of ROS in the SP5C. RESULTS: At the end of the modelling period, chronic migraine mice showed significantly reduced mechanical nociceptive thresholds, as well as photophobic and anxious behaviours. Pretreatment with NBP attenuated nociceptive sensitization, photophobia, and anxiety in the model mice, reduced expression levels of c-Fos and CGRP in the SP5C and activated Nrf2 and its downstream proteins HO-1 and NQO-1. By measuring the associated cytokines, we also found that NBP reduced levels of oxidative stress and inflammation. Most importantly, the therapeutic effect of NBP was significantly reduced after the administration of ML385 to inhibit Nrf2. CONCLUSIONS: Our data suggest that NBP may alleviate migraine by activating the Nrf2 pathway to reduce oxidative stress and inflammation in migraine mouse models, confirming that it may be a potential drug for the treatment of migraine.
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Benzofuranos , Péptido Relacionado con Gen de Calcitonina , Trastornos Migrañosos , Ratones , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/farmacología , Factor 2 Relacionado con NF-E2/uso terapéutico , Enfermedades Neuroinflamatorias , Especies Reactivas de Oxígeno , Fotofobia , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Nitroglicerina/farmacología , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/metabolismoRESUMEN
BACKGROUND: Diarrheal diseases caused by viral agents have led to a great morbidity, mortality, and economic loss in global pig industry. Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and group A porcine rotavirus (RVA) are main causative agents of swine viral diarrhea with similar clinical signs on Chinese farms and their co-infection is also common. However, it is still lack of a convenient method to detect these four agents. METHODS: A TaqMan multiplex qPCR method was developed to detect PEDV, TGEV, PDCoV, and RVA, simultaneously. This method was then applied to investigate 7,342 swine fecal samples or rectal swabs, as well as 1,246 swine intestinal samples collected from 2075 farms in China in 2022. RESULTS: Minimum detection limits of this method were 3 copies/µL for PEDV, 4 copies/µL for TGEV, 8 copies/µL for RVA, and 8 copies/µL for PDCoV, suggesting a good sensitivity. No signals were observed by using this method detecting other viral agents commonly prevalent in pigs, which is suggestive of a good specificity. Application of this method on investigating clinical samples demonstrated a relatively high positive rate for PEDV (22.21%, 1907/8588) and RVA (44.00%, 3779/8588). In addition, co-infection between PEDV and RVA was observed on 360 investigated farms, accounting for 17.35% (360/2075) of the farms where co-infection events were screened. CONCLUSIONS: A TaqMan multiplex qPCR method targeting PEDV, TGEV, PDCoV, and RVA was developed in this study. This method demonstrated a good specificity and sensitivity on investigating these four common viruses responsible for viral diarrhea on Chinese pig farms, which represents a convenient method for the monitoring and differential diagnosis of swine viral diarrhea.
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BACKGROUND: Energy metabolism disorders and neurogenic inflammation play important roles in the central sensitization to chronic migraine (CM). AMP-activated protein kinase (AMPK) is an intracellular energy sensor, and its activation regulates inflammation and reduces neuropathic pain. However, studies on the involvement of AMPK in the regulation of CM are currently lacking. Therefore, this study aimed to explore the mechanism underlying the involvement of AMPK in the central sensitization to CM. METHODS: Mice with recurrent nitroglycerin (NTG)-induced CM were used to detect the expression of AMPK protein in the trigeminal nucleus caudalis (TNC). Following intraperitoneal injection of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and inhibitor compound C, the mechanical pain threshold, activity level, and pain-like behaviors in the mice were measured. The expression of calcitonin gene-related peptide (CGRP) and cytokines, M1/M2 microglia, and NF-κB pathway activation were detected after the intervention. RESULTS: Repeated NTG injections resulted in a gradual decrease in AMPK protein expression, and the negative regulation of AMPK by increased ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression may counteract AMPK activation by increasing ADP/ATP. AICAR can reduce the hyperalgesia and pain-like behaviors of CM mice, improve the activity of mice, reduce the expression of CGRP, IL-1ß, IL-6, and TNF-α in the TNC region, and increase the expression of IL-4 and IL-10. Moreover, AMPK in TNC was mainly located in microglia. AICAR could reduce the expression of inducible NO synthase (iNOS) in M1 microglia and increase the expression of Arginase 1 (Arg1) in M2 microglia by inhibiting the activation of NF-κB pathway. CONCLUSIONS: AMPK was involved in the central sensitization of CM, and the activation of AMPK reduced neuroinflammation in NTG-induced CM mice. AMPK may provide new insights into interventions for energy metabolism disorders and neurogenic inflammation in migraine.
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Trastornos Migrañosos , Nitroglicerina , Ratones , Animales , Nitroglicerina/efectos adversos , Microglía/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , FN-kappa B/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Sensibilización del Sistema Nervioso Central/fisiología , Inflamación Neurogénica/metabolismo , Dolor/metabolismo , Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/metabolismoRESUMEN
Coinfection with multiple viruses is a common phenomenon in clinical settings and is a crucial driver of viral evolution. Although numerous studies have demonstrated viral recombination arising from coinfections of different strains of a specific species, the role of coinfections of different species or genera during viral evolution is rarely investigated. Here, we analyzed coinfections of and recombination events between four different swine enteric coronaviruses that infect the jejunum and ileum in pigs, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), and a deltacoronavirus, porcine deltacoronavirus (PDCoV). Various coinfection patterns were observed in 4,468 fecal and intestinal tissue samples collected from pigs in a 4-year survey. PEDV/PDCoV was the most frequent coinfection. However, recombination analyses have only detected events involving PEDV/TGEV and SADS-CoV/TGEV, indicating that inter-species recombination among coronaviruses is most likely to occur within the same genus. We also analyzed recombination events within the newly identified genus Deltacoronavirus and found that sparrows have played a unique host role in the recombination history of the deltacoronaviruses. The emerging virus PDCoV, which can infect humans, has a different recombination history. In summary, our study demonstrates that swine enteric coronaviruses are a valuable model for investigating the relationship between viral coinfection and recombination, which provide new insights into both inter- and intraspecies recombination events among swine enteric coronaviruses, and extend our understanding of the relationship between coronavirus coinfection and recombination.
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Alphacoronavirus , Coinfección , Infecciones por Coronavirus , Coronavirus , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Virus de la Gastroenteritis Transmisible , Humanos , Porcinos , Animales , Coinfección/veterinaria , Infecciones por Coronavirus/veterinaria , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Gastroenteritis Transmisible/genética , Recombinación GenéticaRESUMEN
The Warburg effect, also referred as aerobic glycolysis, is a common metabolic program during viral infection. Through targeted metabolomics combined with biochemical experiments and various cell models, we investigated the central carbon metabolism (CCM) profiles of cells infected with porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. We found that PDCoV infection required glycolysis but decreased glycolytic flux, exhibiting a non-Warburg effect characterized by pyruvic acid accumulation. Mechanistically, PDCoV enhanced pyruvate kinase activity to promote pyruvic acid anabolism, a process that generates pyruvic acid with concomitant ATP production. PDCoV also hijacked pyruvic acid catabolism to increase biosynthesis of non-essential amino acids (NEAAs), suggesting that pyruvic acid is an essential hub for PDCoV to scavenge host energy and metabolites. Furthermore, PDCoV facilitated glutaminolysis to promote the synthesis of NEAA and pyrimidines for optimal proliferation. Our work supports a novel CCM model after viral infection and provides potential anti-PDCoV drug targets.
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Infecciones por Coronavirus , Coronavirus , Enfermedades de los Porcinos , Porcinos , Animales , Coronavirus/metabolismo , Ácido Pirúvico/metabolismo , Enfermedades de los Porcinos/metabolismo , Enfermedades de los Porcinos/patología , Infecciones por Coronavirus/patologíaRESUMEN
Porcine reproductive and respiratory syndrome (PRRS), which has posed substantial threats to the swine industry worldwide, is primarily characterized by interstitial pneumonia. A disintegrin and metalloproteinase 17 (ADAM17) is a multifunctional sheddase involved in various inflammatory diseases. Herein, our study showed that PRRS virus (PRRSV) infection elevated ADAM17 activity, as demonstrated in primary porcine alveolar macrophages (PAMs), an immortalized PAM cell line (IPAM cells), and the lung tissues of PRRSV-infected piglets. We found that PRRSV infection promoted ADAM17 translocation from the endoplasmic reticulum to the Golgi by enhancing its interaction with inactive rhomboid protein 2 (iRhom2), a newly identified ADAM17 regulator, which in turn elevated ADAM17 activity. By screening for PRRSV-encoded structural proteins, viral envelope (E) and nucleocapsid (N) proteins were identified as the predominant ADAM17 activators. E and N proteins bind with both ADAM17 and iRhom2 to form ternary protein complexes, ultimately strengthening their interactions. Additionally, we demonstrated, using an ADAM17-knockout cell line, that ADAM17 augmented the shedding of soluble TNF-α, a pivotal inflammatory mediator. We also discovered that ADAM17-mediated cleavage of porcine TNF-α occurred between Arg-78 and Ser-79. By constructing a precision mutant cell line with Arg-78-Glu/Ser-79-Glu substitution mutations in TNF-α, we further revealed that the ADAM17-mediated production of soluble TNF-α contributed to the induction of inflammatory responses by PRRSV and its E and N proteins. Taken together, our results elucidate the mechanism by which PRRSV infection activates the iRhom2/ADAM17/TNF-α axis to enhance inflammatory responses, providing valuable insights into the elucidation of PRRSV pathogenesis.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Enfermedades de los Porcinos , Porcinos , Animales , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Pulmón , Macrófagos AlveolaresRESUMEN
The microtubule (MT) is a highly dynamic polymer that functions in various cellular processes through MT hyperacetylation. Thus, many viruses have evolved mechanisms to hijack the MT network of the cytoskeleton to allow intracellular replication of viral genomic material. Coronavirus non-structural protein 8 (nsp8), a component of the viral replication transcriptional complex, is essential for viral survival. Here, we found that nsp8 of porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with a zoonotic potential, inhibits interferon (IFN)-ß production by targeting melanoma differentiation gene 5 (MDA5), the main pattern recognition receptor for coronaviruses in the cytoplasm. Mechanistically, PDCoV nsp8 interacted with MDA5 and induced autophagy to degrade MDA5 in wild-type cells, but not in autophagy-related (ATG)5 or ATG7 knockout cells. Further screening for autophagic degradation receptors revealed that nsp8 interacts with sequestosome 1/p62 and promotes p62-mediated selective autophagy to degrade MDA5. Importantly, PDCoV nsp8 induced hyperacetylation of MTs, which in turn triggered selective autophagic degradation of MDA5 and subsequent inhibition of IFN-ß production. Overall, our study uncovers a novel mechanism employed by PDCoV nsp8 to evade host innate immune defenses. These findings offer new insights into the interplay among viruses, IFNs, and MTs, providing a promising target to develop anti-viral drugs against PDCoV.IMPORTANCECoronavirus nsp8, a component of the viral replication transcriptional complex, is well conserved and plays a crucial role in viral replication. Exploration of the role mechanism of nsp8 is conducive to the understanding of viral pathogenesis and development of anti-viral strategies against coronavirus. Here, we found that nsp8 of PDCoV, an emerging enteropathogenic coronavirus with a zoonotic potential, is an interferon antagonist. Further studies showed that PDCoV nsp8 interacted with MDA5 and sequestosome 1/p62, promoting p62-mediated selective autophagy to degrade MDA5. We further found that PDCoV nsp8 could induce hyperacetylation of MT, therefore triggering selective autophagic degradation of MDA5 and inhibiting IFN-ß production. These findings reveal a novel immune evasion strategy used by PDCoV nsp8 and provide insights into potential therapeutic interventions.
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Infecciones por Coronavirus , Deltacoronavirus , Enfermedades de los Porcinos , Animales , Autofagia , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Deltacoronavirus/metabolismo , Interferones/metabolismo , Microtúbulos/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Porcinos , Enfermedades de los Porcinos/virologíaRESUMEN
HDAC6, a structurally and functionally unique member of the histone deacetylase (HDAC) family, is an important host factor that restricts viral infection. The broad-spectrum antiviral activity of HDAC6 makes it a potent antiviral agent. Previously, we found that HDAC6 functions to antagonize porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. However, the final outcome is typically a productive infection that materializes as cells succumb to viral infection, indicating that the virus has evolved sophisticated mechanisms to combat the antiviral effect of HDAC6. Here, we demonstrate that PDCoV nonstructural protein 5 (nsp5) can cleave HDAC6 at glutamine 519 (Q519), and cleavage of HDAC6 was also detected in the context of PDCoV infection. More importantly, the anti-PDCoV activity of HDAC6 was damaged by nsp5 cleavage. Mechanistically, the cleaved HDAC6 fragments (amino acids 1-519 and 520-1159) lost the ability to degrade PDCoV nsp8 due to their impaired deacetylase activity. Furthermore, nsp5-mediated cleavage impaired the ability of HDAC6 to activate RIG-I-mediated interferon responses. We also tested three other swine enteric coronaviruses (transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome-coronavirus) and found that all these coronaviruses have adopted similar mechanisms to cleave HDAC6 in both an overexpression system and virus-infected cells, suggesting that cleavage of HDAC6 is a common strategy utilized by swine enteric coronaviruses to antagonize the host's antiviral capacity. Together, these data illustrate how swine enteric coronaviruses antagonize the antiviral function of HDAC6 to maintain their infection, providing new insights to the interaction between virus and host.IMPORTANCEViral infections and host defenses are in constant opposition. Once viruses combat or evade host restriction, productive infection is achieved. HDAC6 is a broad-spectrum antiviral protein that has been demonstrated to inhibit many viruses, including porcine deltacoronavirus (PDCoV). However, whether HDAC6 is reciprocally targeted and disabled by viruses remains unclear. In this study, we used PDCoV as a model and found that HDAC6 is targeted and cleaved by nsp5, a viral 3C-like protease. The cleaved HDAC6 loses its deacetylase activity as well as its ability to degrade viral proteins and activate interferon responses. Furthermore, this cleavage mechanism is shared among other swine enteric coronaviruses. These findings shed light on the intricate interplay between viruses and HDAC6, highlighting the strategies employed by viruses to evade host antiviral defenses.
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Infecciones por Coronavirus , Coronavirus , Enfermedades de los Porcinos , Animales , Coronavirus/fisiología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Deltacoronavirus , Interferones/metabolismo , Porcinos , Enfermedades de los Porcinos/virologíaRESUMEN
Lactate, traditionally considered a metabolic by-product, has recently been identified as a substrate for the induction of lactylation, a newly identified epigenetic modification that plays an important role in the regulation of host gene expression. Our previous study showed that lactate levels were significantly elevated in cells infected with the porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide for over 30 years. However, the role of elevated lactate in PRRSV infections remains unknown. In this study, we found that lactate was required for optimal PRRSV proliferation, and PRRSV infection increased cellular lactylation in a dose-dependent manner. Using the Cleavage Under Targets and Tagmentation (CUT&Tag) combined with RNA sequencing (RNA-seq) to screen the downstream genes regulated by lactylation in PRRSV-infected cells, we found that PRRSV-induced lactylation activated the expression of heat shock 70 kDa protein 6 (HSPA6). Follow-up experiments showed that HSPA6 is important for PRRSV proliferation by negatively modulating interferon (IFN)-ß induction. Mechanistically, HSPA6 impeded the interaction between TNF-receptor-associated factor 3 (TRAF3) and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε), thereby hindering the production of IFN-ß. Taken together, these results indicate that the activated lactate-lactylation-HSPA6 axis promotes viral growth by impairing IFN-ß induction, providing new therapeutic targets for the prevention and control of PRRSV infection. The results presented here also link lactylation to the virus life cycle, improving our understanding of epigenetic regulation in viral infection.IMPORTANCEAs a newly identified epigenetic modification, lactate-induced lactylation has received attentions because it plays important roles in gene expression and contributes to tumorigenesis and the innate immune response. Previous studies showed that many viruses upregulate cellular lactate levels; however, whether virus-elevated lactate induces lactylation and the subsequent biological significance of the modification to viral infection have not been reported. In this study, we demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection induced cellular lactylation, which, in turn, upregulated the expression of HSPA6, an IFN-negative regulator. We also dissected the mechanism by which HSPA6 negatively regulates IFN-ß production. To our knowledge, this is the first report to study virus-induced lactylation and establish the relationship between lactylation and virus infection.
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Ácido Láctico , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Epigénesis Genética , Expresión Génica , Ácido Láctico/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Porcinos , Replicación ViralRESUMEN
Chiral nanoparticles (C-NPs) play a crucial role in biomedical applications, especially in their biological effects on cytotoxicity and metabolism. However, there are rare reports about the antivirus property of C-NPs and their working mechanism. Here, three different types of chiral ZnO NPs (l-ZnO, d-ZnO, and dl-ZnO) were prepared as enantioselective antivirals. Biocompatibility test results showed that the three different chiral ZnO NPs varied significantly in cytotoxicity. Evaluation of their effects against porcine reproductive and respiratory syndrome virus (PRRSV) indicated that compared with d-ZnO and dl-ZnO NPs, l-ZnO NPs exhibited stronger anti-PRRSV activity due to their higher cognate cell adhesion and uptake. Furthermore, the high concentration of l-ZnO NPs can obviously reduce cellular reactive oxygen species (ROS) in MARC-145 cells, thus effectively preventing PRRSV-induced oxidative damage. This study demonstrated the outstanding antiviral properties of l-ZnO NPs, which may facilitate the development and application of C-NPs in antiviral drugs and tissue engineering.
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Nanopartículas del Metal , Nanopartículas , Óxido de Zinc , Óxido de Zinc/farmacología , Estereoisomerismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Antivirales/farmacologíaRESUMEN
Clinical imaging studies have revealed that the hypothalamus is activated in migraine patients prior to the onset of and during headache and have also shown that the hypothalamus has increased functional connectivity with the spinal trigeminal nucleus. The dopaminergic system of the hypothalamus plays an important role, and the dopamine-rich A11 nucleus may play an important role in migraine pathogenesis. We used intraperitoneal injections of glyceryl trinitrate to establish a model of acute migraine attack and chronicity in mice, which was verified by photophobia experiments and von Frey experiments. We explored the A11 nucleus and its downstream pathway using immunohistochemical staining and neuronal tracing techniques. During acute migraine attack and chronification, c-fos expression in GABAergic neurons in the A11 nucleus was significantly increased, and inhibition of DA neurons was achieved by binding to GABA A-type receptors on the surface of dopaminergic neurons in the A11 nucleus. However, the expression of tyrosine hydroxylase and glutamic acid decarboxylase proteins in the A11 nucleus of the hypothalamus did not change significantly. Specific destruction of dopaminergic neurons in the A11 nucleus of mice resulted in severe nociceptive sensitization and photophobic behavior. The expression levels of the D1 dopamine receptor and D2 dopamine receptor in the caudal part of the spinal trigeminal nucleus candalis of the chronic migraine model were increased. Skin nociceptive sensitization of mice was slowed by activation of the D2 dopamine receptor in SP5C, and activation of the D1 dopamine receptor reversed this behavioral change. GABAergic neurons in the A11 nucleus were activated and exerted postsynaptic inhibitory effects, which led to a decrease in the amount of DA secreted by the A11 nucleus in the spinal trigeminal nucleus candalis. The reduced DA bound preferentially to the D2 dopamine receptor, thus exerting a defensive effect against headache.
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Dopamina , Trastornos Migrañosos , Ratones , Humanos , Animales , Dopamina/metabolismo , Núcleo Espinal del Trigémino/metabolismo , Hipotálamo/metabolismo , Receptores de Dopamina D1/metabolismo , Trastornos Migrañosos/metabolismo , Neuronas Dopaminérgicas/metabolismo , Cefalea/metabolismoRESUMEN
IMPORTANCE: Understanding the role of the endoribonuclease non-structural protein 15 (nsp15) (EndoU) in coronavirus (CoV) infection and pathogenesis is essential for vaccine target discovery. Whether the EndoU activity of CoV nsp15, as a virulence-related protein, has a diverse effect on viral virulence needs to be further explored. Here, we found that the transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) nsp15 proteins antagonize SeV-induced interferon-ß (IFN-ß) production in human embryonic kidney 293 cells. Interestingly, compared with wild-type infection, infection with EnUmt-TGEV or EnUmt-FIPV did not change the IFN-ß response or reduce viral propagation in immunocompetent cells. The results of animal experiments showed that EnUmt viruses did not reduce the clinical presentation and mortality caused by TGEV and FIPV. Our findings enrich the understanding of nsp15-mediated regulation of alpha-CoV propagation and virulence and reveal that the conserved functions of nonstructural proteins have diverse effects on the pathogenicity of CoVs.
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Infecciones por Coronavirus , Coronavirus , Animales , Humanos , Virulencia , Endorribonucleasas/metabolismo , Endorribonucleasas Específicas de UridilatoRESUMEN
IMPORTANCE: Retrograde transport has been reported to be closely associated with normal cellular biological processes and viral replication. As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has attracted considerable attention. However, whether retrograde transport is associated with PDCoV infection remains unclear. Our present study demonstrates that retromer protein VPS35 acts as a critical host factor that is required for PDCoV infection. Mechanically, VPS35 interacts with PDCoV NS6, mediating the retrograde transport of NS6 from endosomes to the Golgi and preventing it from lysosomal degradation. Recombinant PDCoVs with an NS6 deletion display resistance to VPS35 deficiency. Our work reveals a novel evasion mechanism of PDCoV that involves the manipulation of the retrograde transport pathway by VPS35, providing new insight into the mechanism of PDCoV infection.