RESUMEN
Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection.
Asunto(s)
Alphacoronavirus , Proteínas de Unión al Calcio , Virus de la Diarrea Epidémica Porcina , Animales , Alphacoronavirus/metabolismo , Línea Celular , Chlorocebus aethiops , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células Epiteliales/metabolismo , Virus de la Diarrea Epidémica Porcina/metabolismo , Porcinos , Células Vero , Replicación Viral , Proteínas de Unión al Calcio/metabolismoRESUMEN
African swine fever virus (ASFV) causes severe disease in domestic pigs and wild boars, seriously threatening the development of the global pig industry. Type I interferon (IFN-I) is an important component of innate immunity, inducing the transcription and expression of antiviral cytokines by activating Janus-activated kinase-signal transducer and activator of transcription (STAT). However, the underlying molecular mechanisms by which ASFV antagonizes IFN-I signaling have not been fully elucidated. Therefore, using coimmunoprecipitation, confocal microscopy, and dual luciferase reporter assay methods, we investigated these mechanisms and identified a novel ASFV immunosuppressive protein, pB475L, which interacts with the C-terminal domain of STAT2. Consequently, pB475L inhibited IFN-I signaling by inhibiting STAT1 and STAT2 heterodimerization and nuclear translocation. Furthermore, we constructed an ASFV-B475L7PM mutant strain by homologous recombination, finding that ASFV-B475L7PM attenuated the inhibitory effects on IFN-I signaling compared to ASFV-WT. In summary, this study reveals a new mechanism by which ASFV impairs host innate immunity.
Asunto(s)
Virus de la Fiebre Porcina Africana , Inmunidad Innata , Interferón Tipo I , Factor de Transcripción STAT2 , Transducción de Señal , Proteínas Virales , Animales , Humanos , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/genética , Células HEK293 , Evasión Inmune , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT2/metabolismo , Factor de Transcripción STAT2/genética , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/inmunologíaRESUMEN
IMPORTANCE: Porcine epidemic diarrhea, characterized by vomiting, dehydration, and diarrhea, is an acute and highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV) in neonatal piglets. This disease has caused large economic losses to the porcine industry worldwide. Thus, identifying the host factors involved in PEDV infection is important to develop novel strategies to control PEDV transmission. This study shows that PEDV infection upregulates karyopherin α 2 (KPNA2) expression in Vero and intestinal epithelial (IEC) cells. KPNA2 binds to and degrades the PEDV E protein via autophagy to suppress PEDV replication. These results suggest that KPNA2 plays an antiviral role against PEDV. Specifically, knockdown of endogenous KPNA2 enhances PEDV replication, whereas its overexpression inhibits PEDV replication. Our data provide novel KPNA2-mediated viral restriction mechanisms in which KPNA2 suppresses PEDV replication by targeting and degrading the viral E protein through autophagy. These mechanisms can be targeted in future studies to develop novel strategies to control PEDV infection.
Asunto(s)
Autofagia , Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Animales , Chlorocebus aethiops , Infecciones por Coronavirus/veterinaria , Diarrea/veterinaria , Virus de la Diarrea Epidémica Porcina/fisiología , Porcinos , Enfermedades de los Porcinos , Células Vero , Proteínas del Envoltorio Viral , Proteínas Virales , Replicación ViralRESUMEN
African swine fever (ASF) is a highly infectious disease caused by the African swine fever virus (ASFV) in swine. It is characterized by the death of cells in infected tissues. However, the molecular mechanism of ASFV-induced cell death in porcine alveolar macrophages (PAMs) remains largely unknown. In this study, transcriptome sequencing of ASFV-infected PAMs found that ASFV activated the JAK2-STAT3 pathway in the early stages and apoptosis in the late stages of infection. Meanwhile, the JAK2-STAT3 pathway was confirmed to be essential for ASFV replication. AG490 and andrographolide (AND) inhibited the JAK2-STAT3 pathway, promoted ASFV-induced apoptosis, and exerted antiviral effects. Additionally, CD2v promoted STAT3 transcription and phosphorylation as well as translocation into the nucleus. CD2v is the main envelope glycoprotein of the ASFV, and further investigations showed that CD2v deletion downregulates the JAK2-STAT3 pathway and promotes apoptosis to inhibit ASFV replication. Furthermore, we discovered that CD2v interacts with CSF2RA, which is a hematopoietic receptor superfamily member in myeloid cells and a key receptor protein that activates receptor-associated JAK and STAT proteins. In this study, CSF2RA small interfering RNA (siRNA) downregulated the JAK2-STAT3 pathway and promoted apoptosis to inhibit ASFV replication. Taken together, ASFV replication requires the JAK2-STAT3 pathway, while CD2v interacts with CSF2RA to regulate the JAK2-STAT3 pathway and inhibit apoptosis to facilitate virus replication. These results provide a theoretical basis for the escape mechanism and pathogenesis of ASFV. IMPORTANCE African swine fever is a hemorrhagic disease caused by the African swine fever virus (ASFV), which infects pigs of different breeds and ages, with a fatality rate of up to 100%. It is one of the key diseases affecting the global livestock industry. Currently, no commercial vaccines or antiviral drugs are available. Here, we show that ASFV replicates via the JAK2-STAT3 pathway. More specifically, ASFV CD2v interacts with CSF2RA to activate the JAK2-STAT3 pathway and inhibit apoptosis, thereby maintaining the survival of infected cells and promoting viral replication. This study revealed an important implication of the JAK2-STAT3 pathway in ASFV infection and identified a novel mechanism by which CD2v has evolved to interact with CSF2RA and maintain JAK2-STAT3 pathway activation to inhibit apoptosis, thus elucidating new information regarding the signal reprogramming of host cells by ASFV.
Asunto(s)
Virus de la Fiebre Porcina Africana , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos , Proteínas del Envoltorio Viral , Replicación Viral , Animales , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Apoptosis/genética , Porcinos , Replicación Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Interacciones Microbiota-Huesped , Regulación hacia AbajoRESUMEN
African swine fever (ASF) is an acute and severe infectious disease caused by the ASF virus (ASFV). The mortality rate of ASF in pigs can reach 100%, causing huge economic losses to the pig industry. Here, we found that ASFV protein MGF505-7R inhibited the beta interferon (IFN-ß)-mediated Janus-activated kinase-signal transducer and activation of transcription (JAK-STAT) signaling. Our results demonstrate that MGF505-7R inhibited interferon-stimulated gene factor 3 (ISGF3)-mediated IFN-stimulated response element (ISRE) promoter activity. Importantly, we observed that MGF505-7R inhibits ISGF3 heterotrimer formation by interacting with interferon regulatory factor 9 (IRF9) and inhibits the nuclear translocation of ISGF3. Moreover, to demonstrate the role of MGF505-7R in IFN-I signal transduction during ASFV infection, we constructed and evaluated ASFV-ΔMGF505-7R recombinant viruses. ASFV-ΔMGF505-7R restored STAT2 and STAT1 phosphorylation, alleviated the inhibition of ISGF3 nuclear translocation, and showed increased susceptibility to IFN-ß, unlike the parental GZ201801 strain. In conclusion, our study shows that ASFV protein MGF505-7R plays a key role in evading IFN-I-mediated innate immunity, revealing a new mode of evasion for ASFV. IMPORTANCE ASF, caused by ASFV, is currently prevalent in Eurasia, with mortality rates reaching 100% in pigs. At present, there are no safe or effective vaccines against ASFV. In this study, we found that the ASFV protein MGF505-7R hinders IFN-ß signaling by interacting with IRF9 and inhibiting the formation of ISGF3 heterotrimers. Of note, we demonstrated that MGF505-7R plays a role in the immune evasion of ASFV in infected hosts and that recombinant viruses alleviated the effect on type I IFN (IFN-I) signaling and exhibited increased susceptibility to IFN-ß. This study provides a theoretical basis for developing vaccines against ASFV using strains with MGF505-7R gene deletions.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Interferón Tipo I , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón , Replicación Viral , Animales , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Inmunidad Innata , Interferón Tipo I/inmunología , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/inmunología , Transducción de Señal , Porcinos , Proteínas Virales/genética , Proteínas Virales/inmunología , Replicación Viral/fisiología , Transporte Activo de Núcleo Celular/genética , Evasión Inmune/genéticaRESUMEN
Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its broad cellular tropism suggests a potential risk of cross-species transmission. A limited understanding of PEAV entry mechanisms may hinder a rapid response to potential outbreaks. This study analyzed PEAV entry events using chemical inhibitors, RNA interference, and dominant-negative mutants. PEAV entry into Vero cells depended on three endocytic pathways: caveolae, clathrin, and macropinocytosis. Endocytosis requires dynamin, cholesterol, and a low pH. Rab5, Rab7, and Rab9 GTPases (but not Rab11) regulate PEAV endocytosis. PEAV particles colocalize with EEA1, Rab5, Rab7, Rab9, and Lamp-1, suggesting that PEAV translocates into early endosomes after internalization, and Rab5, Rab7, and Rab9 regulate trafficking to lysosomes before viral genome release. PEAV enters porcine intestinal cells (IPI-2I) through the same endocytic pathway, suggesting that PEAV may enter various cells through multiple endocytic pathways. This study provides new insights into the PEAV life cycle. IMPORTANCE Emerging and reemerging coronaviruses cause severe human and animal epidemics worldwide. PEAV is the first bat-like coronavirus to cause infection in domestic animals. However, the PEAV entry mechanism into host cells remains unknown. This study demonstrates that PEAV enters into Vero or IPI-2I cells through caveola/clathrin-mediated endocytosis and macropinocytosis, which does not require a specific receptor. Subsequently, Rab5, Rab7, and Rab9 regulate PEAV trafficking from early endosomes to lysosomes, which is pH dependent. The results advance our understanding of the disease and help to develop potential new drug targets against PEAV.
Asunto(s)
Alphacoronavirus , Caveolas , Clatrina , Pinocitosis , Internalización del Virus , Proteínas de Unión al GTP rab , Alphacoronavirus/fisiología , Proteínas de Unión al GTP rab/metabolismo , Endosomas/metabolismo , Infecciones por Coronavirus/metabolismo , Concentración de Iones de Hidrógeno , Dinaminas/metabolismo , Caveolas/metabolismo , Colesterol/metabolismo , Clatrina/metabolismo , Pinocitosis/fisiología , Células Vero , Chlorocebus aethiops , AnimalesRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease in pigs of all ages and reproductive failure in sows, resulting in great economic losses to the swine industry. In this work, we identified the interaction between PSMB4 and PRRSV Nsp1α by yeast two-hybrid screening. The PSMB4-Nsp1α interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and laser confocal experiments. The PCPα domain (amino acids 66 to 166) of Nsp1α and the C-terminal domain (amino acids 250 to 264) of PSMB4 were shown to be critical for the PSMB4-Nsp1α interaction. PSMB4 overexpression reduced PRRSV replication, whereas PSMB4 knockdown elicited opposing effects. Mechanistically, PSMB4 targeted K169 in Nsp1α for K63-linked ubiquitination and targeted Nsp1α for autolysosomal degradation by interacting with LC3 to enhance the activation of the lysosomal pathway. Meanwhile, we found that PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. In conclusion, our data revealed a new mechanism of PSMB4-mediated restriction of PRRSV replication, whereby PSMB4 was found to induce Nsp1α degradation and type I interferon expression, in order to impede the replication of PRRSV. IMPORTANCE In the swine industry, PRRSV is a continuous threat, and the current vaccines are not effective enough to block it. This study determined that PSMB4 plays an antiviral role against PRRSV. PSMB4 was found to interact with PRRSV Nsp1α, mediate K63-linked ubiquitination of Nsp1α at K169, and thus trigger its degradation via the lysosomal pathway. Additionally, PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. This study extends our understanding of the proteasome subunit PSMB4 against PRRSV replication and will contribute to the development of new antiviral strategies.
Asunto(s)
Interferón Tipo I , Virus del Síndrome Respiratorio y Reproductivo Porcino , Complejo de la Endopetidasa Proteasomal , Proteínas no Estructurales Virales , Expresión Génica/inmunología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interferón beta/genética , Lisosomas/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/inmunología , Dominios Proteicos , Proteolisis , Porcinos , Ubiquitinación , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genética , AnimalesRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral pathogen with substantial economic implications for the global swine industry. The existing vaccination strategies and antiviral drugs offer limited protection. Replication of the viral RNA genome encompasses a complex series of steps, wherein a replication complex is assembled from various components derived from both viral and cellular sources, as well as from the viral genomic RNA template. In this study, we found that ZNF283, a Krüppel-associated box (KRAB) containing zinc finger protein, was upregulated in PRRSV-infected Marc-145 cells and porcine alveolar macrophages and that ZNF283 inhibited PRRSV replication and RNA synthesis. We also found that ZNF283 interacts with the viral proteins Nsp9, an RNA-dependent RNA polymerase, and Nsp10, a helicase. The main regions involved in the interaction between ZNF283 and Nsp9 were determined to be the KRAB domain of ZNF283 and amino acids 178-449 of Nsp9. The KRAB domain of ZNF283 plays a role in facilitating Nsp10 binding. In addition, ZNF283 may have an affinity for the 3' untranslated region of PRRSV. These findings suggest that ZNF283 is an antiviral factor that inhibits PRRSV infection and extend our understanding of the interactions between KRAB-containing zinc finger proteins and viruses.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Enfermedades de los Porcinos , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Unión Proteica , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , ARN Viral/metabolismo , Dedos de Zinc , Replicación ViralRESUMEN
The site-selective reaction of substrates with multiple reactive sites has been a focus of the current synthetic chemistry. The use of attractive noncovalent interactions between the catalyst and substrate is emerging as a versatile approach to address site-selectivity challenges. Herein, we designed and synthesized a series of palladacycles, to control meta-selective Suzuki coupling of 3,4-dichlorophenol and 3,4-dichlorobenzyl alcohol. Noncovalent interactions directed zwitterionic aqua palladacycles catalyzed meta-selective Suzuki couplings of 3,4-dichloroarenes bearing hydroxyl in water have been developed. Experiments and density functional theory (DFT) calculations demonstrated that the electrostatic interactions play a critical role in meta-selective coupling of 3,4-dichlorophenol, while meta-selective coupling of 3,4-dichlorobenzyl alcohol arises due to the hydrogen-bonding interactions.
RESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide. Currently, vaccine strategies provide limited protection against PRRSV transmission, and no effective drug is commercially available. Therefore, there is an urgent need to develop novel antiviral strategies to prevent PRRSV pandemics. This study showed that artesunate (AS), one of the antimalarial drugs, potently suppressed PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs) at micromolar concentrations. Furthermore, we demonstrated that this suppression was closely associated with AS-activated AMPK (energy homeostasis) and Nrf2/HO-1 (inflammation) signaling pathways. AS treatment promoted p-AMPK, Nrf2, and HO-1 expression and, thus, inhibited PRRSV replication in Marc-145 and PAM cells in a time- and dose-dependent manner. These effects of AS were reversed when the AMPK or HO-1 gene was silenced by short interfering RNA. In addition, we demonstrated that AMPK works upstream of Nrf2/HO-1, as its activation by AS is AMPK dependent. Adenosine phosphate analysis showed that AS activates AMPK via improving the AMP/ADP-to-ATP ratio rather than direct interaction with AMPK. Altogether, our findings indicate that AS is a promising novel therapeutic for controlling PRRSV and that its anti-PRRSV mechanism, which involves the functional link between energy homeostasis and inflammation suppression pathways, may provide opportunities for developing novel antiviral agents. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) infections have continuously threatened the pork industry worldwide. Vaccination strategies provide very limited protection against PRRSV infection, and no effective drug is commercially available. We show that artesunate (AS), one of the antimalarial drugs, is a potent inhibitor against PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs). Furthermore, we demonstrate that AS inhibits PRRSV replication via activation of AMPK-dependent Nrf2/HO-1 signaling pathways, revealing a novel link between energy homeostasis (AMPK) and inflammation suppression (Nrf2/HO-1) during viral infection. Therefore, we believe that AS may be a promising novel therapeutics for controlling PRRSV, and its anti-PRRSV mechanism may provide a strategy to develop novel antiviral agents.
Asunto(s)
Antimaláricos/farmacología , Artesunato/farmacología , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Transducción de Señal/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antimaláricos/química , Artesunato/química , Línea Celular , Susceptibilidad a Enfermedades , Hemo-Oxigenasa 1/metabolismo , Interacciones Huésped-Patógeno , Modelos Biológicos , Factor 2 Relacionado con NF-E2/metabolismo , PorcinosRESUMEN
African swine fever (ASF) is an acute infectious haemorrhagic fever of pigs caused by African swine fever virus (ASFV). Aloe-emodin (Ae) is an active ingredient of Chinese herbs with antiviral, anticancer, and anti-inflammatory effects. We investigated the antiviral activity and mechanism of action of Ae against ASFV using Real-time quantitative PCR (qPCR), western blotting, and indirect immunofluorescence assays. Ae significantly inhibited ASFV replication. Furthermore, transcriptomic analysis revealed that ASFV infection activated the NF-κB signaling pathway in the early stage and the apoptosis pathway in the late stage. Ae significantly downregulated the expression levels of MyD88, phosphor-NF-κB p65, and pIκB proteins as well as the mRNA levels of IL-1ß and IL-8 in porcine alveolar macrophages (PAMs) infected with ASFV, thereby inhibiting the activation of the NF-κB signaling pathway induced by ASFV. Flow cytometry and western blot analysis revealed that Ae significantly increased the percentage of ASFV-induced apoptotic cells. Additionally, Ae promoted apoptosis by upregulating the expression levels of cleaved-caspase3 and Bax proteins and downregulating the expression levels of Bcl-2 proteins. This suggests that Ae promotes apoptosis by inhibiting the NF-κB pathway, resulting in inhibition of ASFV replication. These findings have further improved therapeutic reserves for the prevention and treatment of ASF.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Aloe , Emodina , Animales , Virus de la Fiebre Porcina Africana/genética , Aloe/metabolismo , Antivirales/farmacología , Apoptosis , Emodina/farmacología , FN-kappa B/metabolismo , Transducción de Señal , Porcinos , Replicación ViralRESUMEN
BACKGROUND: African swine fever virus (ASFV) is one of the most fatal swine etiological agents and has a huge economic impact on the global pork industry. Given that no effective vaccines or anti-ASFV drugs are available, there remains a pressing need for novel anti-ASFV drugs. This study aimed to investigate the anti-African swine fever virus (ASFV) activity of brequinar, a DHODH inhibitor. METHODS: The anti-ASFV activity of brequinar was investigated using IFA, HAD, HAD50, qRT-PCR, and western blotting assays. The western blotting assay was used to investigate whether brequinar inhibits ASFV replication by killing ASFV particles directly or by acting on cell factors. The confocal microscopy and western blotting assays were used to investigate whether brequinar inhibits ASFV replication by activating ferroptosis. RESULTS: In this study, brequinar was found to effectively inhibit ASFV replication ex vivo in porcine alveolar macrophages (PAMs) in a dose-dependent manner. In kinetic studies, brequinar was found to maintain ASFV inhibition from 24 to 72 hpi. Mechanistically, the time-of-addition assay showed that brequinar exerted anti-ASFV activity in all treatment modes, including pre-, co-, and post-treatment rather than directly killing ASFV particles. Notably, FerroOrange, Mito-FerroGreen, and Liperfluo staining experiments showed that brequinar increased the accumulation of intracellular iron, mitochondrial iron, and lipid peroxides, respectively. Furthermore, we also found that ferroptosis agonist cisplatin treatment inhibited ASFV replication in a dose-dependent manner and the inhibitory effect of brequinar on ASFV was partially reversed by the ferroptosis inhibitor ferrostatin-1, suggesting that brequinar activates ferroptosis to inhibit ASFV replication. Interestingly, exogenous uridine supplementation attenuated the anti-ASFV activity of brequinar, indicating that brequinar inhibits ASFV replication by inhibiting DHODH activity and the depletion of intracellular pyrimidine pools; however, the induction of ferroptosis by brequinar treatment was not reversed by exogenous uridine supplementation, suggesting that brequinar activation of ferroptosis is not related to the metabolic function of pyrimidines. CONCLUSIONS: Our data confirm that brequinar displays potent antiviral activity against ASFV in vitro and reveal the mechanism by which brequinar inhibits ASFV replication by activating ferroptosis, independent of inhibiting pyrimidine synthesis, providing novel targets for the development of anti-ASFV drugs.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Ferroptosis , Porcinos , Animales , Replicación Viral , Dihidroorotato Deshidrogenasa , Cinética , Uridina/metabolismo , Hierro/metabolismoRESUMEN
African swine fever (ASF), caused by ASF virus (ASFV) infection, poses a huge threat to the pork industry owing to ineffective preventive and control measures. Hence, there is an urgent need to develop strategies, including antiviral drugs targeting ASFV, for preventing ASFV spread. This study aimed to identify novel compounds with anti-ASFV activity. To this end, we screened a small chemical library of 102 compounds, among which the natural flavonoid dihydromyricetin (DHM) exhibited the most potent anti-ASFV activity. DHM treatment inhibited ASFV replication in a dose- and time-dependent manner. Furthermore, it inhibited porcine reproductive and respiratory syndrome virus and swine influenza virus replication, which suggested that DHM exerts broad-spectrum antiviral effects. Mechanistically, DHM treatment inhibited ASFV replication in various ways in the time-to-addition assay, including pre-, co-, and post-treatment. Moreover, DHM treatment reduced the levels of ASFV-induced inflammatory mediators by regulating the TLR4/MyD88/MAPK/NF-κB signaling pathway. Meanwhile, DHM treatment reduced the ASFV-induced accumulation of reactive oxygen species, further minimizing pyroptosis by inhibiting the ASFV-induced NLRP3 inflammasome activation. Interestingly, the effects of DHM on ASFV were partly reversed by treatment with polyphyllin VI (a pyroptosis agonist) and RS 09 TFA (a TLR4 agonist), suggesting that DHM inhibits pyroptosis by regulating TLR4 signaling. Furthermore, targeting TLR4 with resatorvid (a specific inhibitor of TLR4) and small interfering RNA against TLR4 impaired ASFV replication. Taken together, these results reveal the anti-ASFV activity of DHM and the underlying mechanism of action, providing a potential compound for developing antiviral drugs targeting ASFV.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Enfermedades de los Porcinos , Animales , Porcinos , Receptor Toll-Like 4 , Piroptosis , Antivirales/farmacologíaRESUMEN
A novel CuS/BaWO4 heterojunction catalyst was prepared and characterized. Taking bisphenol A as the target pollutant for catalytic degradation, the sonocatalytic activity of CuS/BaWO4 composite was evaluated, and the combination with persulfate improved the sonocatalytic degradation of bisphenol A. The results showed that CuS/BaWO4 composite had good sonocatalytic degradation activity for bisphenol A, and the degradation rate was 70.99% ± 1.46%. After combined with persulfate, the degradation rate was further increased to 95.34% ± 0.10%, and the reaction time was relatively shortened. The results of the trapping experiment and calculated energy band positions showed that the formation of S-scheme heterojunction and the formation of hydroxyl radicals and holes were the key to the catalytic degradation of bisphenol A by CuS/BaWO4 composite. In this study, a new CuS/BaWO4 heterojunction sonocatalyst was synthesized. The catalyst can efficiently remove bisphenol A from the water environment and can be used as a potential solution for endocrine disruptor pollution in the water environment.
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Compuestos de Bencidrilo , Ultrasonido , Agua , Compuestos de Bario/química , Catálisis , Compuestos de Tungsteno/químicaRESUMEN
It has been established that Cutaneous T-Cell lymphomas (CTCL) are caused by the monoclonal proliferation of T lymphocytes in the skin. This heterogeneous group of diseases represents a significant source of distress to patients since the diagnosis and treatment are often challenging. As one of the most abundant internal modifications in mRNA in higher eukaryotes, N6-methyladenosine (m6A) is widely recognized to affect the development and progression of cancers. However, knowledge on the involvement of m6A in CTCL is still limited. In this work, we revealed the role of METTL3-mediated m6A modification in CTCL progression. ELISA, western blot, and qRT-PCR assays demonstrated that METTL3 was significantly downregulated in CTCL cells both in vivo and in vitro. CCK-8, EdU, flow cytometry, and transwell assays showed that the decline in METTL3 levels was responsible for CTCL cell proliferation and migration. Furthermore, using small interfering RNAs against METTL3 and the RIP assay, we showed that CDKN2A was a key regulator during this process in vitro and in vivo, and insufficient methylation modification blocked the interaction between CDKN2A and m6A reader IGF2BP2, resulting in mRNA degradation. To the best of our knowledge, this is the first study to depict the role of m6A in CTCL development and provide potential bio-targets for therapy.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina , Linfoma Cutáneo de Células T , Metiltransferasas , Proteínas de Unión al ARN , Adenosina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Humanos , Linfoma Cutáneo de Células T/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND: Finding the key amino acid sites that could affect viral biological properties or protein functions has always been a topic of substantial interest in virology. The nucleocapsid (N) protein is one of the principal proteins of the porcine reproductive and respiratory syndrome virus (PRRSV) and plays a vital role in the virus life cycle. The N protein has only 123 or 128 amino acids, some of key amino acid sites which could affect the protein functions or impair the viral biological characteristics have been identified. In this research, our objective was to find out whether there are other novel amino acid sites of the N protein can affect N protein functions or PRRSV-2 replication. RESULTS: In this study, we found mutated the serine78 and serine 99of the nucleocapsid (N) protein can reduce the N-induced expression of IL-10 mRNA; Then, by using reverse genetics system, we constructed and rescued the mutant viruses, namely, A78 and A99.The IFA result proved that the mutations did not affect the rescue of the PRRSV-2. However, the results of the multistep growth kinetics and qPCR assays indicated that, compared with the viral replication ability, the titres and gRNA levels of A78 were significantly decreased compared with the wild-type. Further study showed that a single amino acid change from serine to alanine at position 78 of the N protein could abrogates the level of viral genomic and subgenomic RNAs. It means the mutation could significant decrease the viral replication efficiency in vitro. CONCLUSIONS: Our results suggest that the serine78 of N protein is a key site which could affect the N protein function and PRRSV replication ability.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Enfermedades de los Porcinos , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Línea Celular , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , ARN Viral/genética , Serina/química , Porcinos , Replicación Viral/fisiologíaRESUMEN
Gold-nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective posthybridization manipulations for genomic analysis. Here, we present a new design for a colorimetric gene-sensing platform based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the trans-ssDNA or -ssRNA cleavage. Target-induced trans-ssDNA or ssRNA cleavage triggers an aggregation behavior change for the designed AuNPs-DNA probes pair, enabling the completion of naked-eye gene detection (transgenic rice, African swine fever virus, and miRNAs as the models) within 1 h. This platform is also showing promise as a fast and inexpensive tool for bacteria identification using 16S rDNA or 16S rRNA. A CRISPR/Cas-based colorimetric platform shows superior characteristics, such as probe universality, compatibility with isothermal reaction conditions, on-site detection capability, and high sensitivity, thus, demonstrating its use as a robust next-generation gene detection platform.
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Sistemas CRISPR-Cas/genética , Colorimetría/métodos , ARN Ribosómico 16S/análisis , Virus de la Fiebre Porcina Africana/genética , Animales , Bacterias/genética , Sondas de ADN/química , ADN Viral/análisis , ADN Viral/química , Oro/química , Nanopartículas del Metal/química , MicroARNs/análisis , MicroARNs/química , Regiones Promotoras Genéticas , ARN Ribosómico 16S/química , PorcinosRESUMEN
BACKGROUND: As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV. RESULTS: Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency. CONCLUSIONS: In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.
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Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/diagnóstico , Sistemas CRISPR-Cas/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Fiebre Porcina Africana/virología , Animales , Proteínas Bacterianas/genética , Sangre/virología , Proteínas Asociadas a CRISPR/genética , Endodesoxirribonucleasas/genética , Técnicas de Diagnóstico Molecular/métodos , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , PorcinosRESUMEN
BACKGROUND: The value of the CT features and quantitative analysis of lung subsolid nodules (SSNs) in the prediction of the pathological grading of lung adenocarcinoma is discussed. METHODS: Clinical data and CT images of 207 cases (216 lesions) with CT manifestations of an SSNs lung adenocarcinoma confirmed by surgery pathology were retrospectively analysed. The pathological results were divided into three groups, including atypical adenomatous hyperplasia (AAH)/adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma (IAC). Then, the quantitative and qualitative data of these nodules were compared and analysed. RESULTS: The mean size, maximum diameter, mean CT value and maximum CT value of the nodules were significantly different among the three groups of AAH/AIS, MIA and IAC and were different between the paired groups (AAH/AIS and MIA or MIA and IAC) (P < 0.05). The critical values of the above indicators between AAH/AIS and MIA were 10.05 mm, 11.16 mm, - 548.00 HU and - 419.74 HU. The critical values of the above indicators between MIA and IAC were 14.42 mm, 16.48 mm, - 364.59 HU and - 16.98 HU. The binary logistic regression analysis of the features with the statistical significance showed that the regression model between AAH/AIS and MIA is logit(p) = - 0.93 + 0.216X1 + 0.004X4. The regression model between MIA and IAC is logit(p) = - 1.242-1.428X5(1) - 1.458X6(1) + 1.146X7(1) + 0.272X2 + 0.005X3. The areas under the curve (AUC) obtained by plotting the receiver operating characteristic curve (ROC) using the regression probabilities of regression models I and II were 0.815 and 0.931. CONCLUSIONS: Preoperative prediction of pathological classification of CT image features has important guiding value for clinical management. Correct diagnosis results can effectively improve the patient survival rate. Through comprehensive analysis of the CT features and qualitative data of SSNs, the diagnostic accuracy of SSNs can be effectively improved. The logistic regression model established in this study can better predict the pathological classification of SSNs lung adenocarcinoma on CT, and the predictive value is significantly higher than the independent use of each quantitative factor.
Asunto(s)
Adenocarcinoma del Pulmón/diagnóstico por imagen , Adenocarcinoma del Pulmón/patología , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estudios RetrospectivosRESUMEN
BACKGROUND: Bufavirus is a newly discovered zoonotic virus reported in numerous mammals and humans. However, the epidemiological and genetic characteristics of porcine bufaviruses (PBuVs) in China remain unclear. METHODS: To detect PBuVs in China, 384 samples (92 fecal and 292 serum specimens) were collected from 2017 to 2018, covering six provinces in China, and were evaluated by nested PCR. Further, the positive samples from different provinces were selected to obtain the complete genome of Chinese PBuVs. RESULTS: The prevalence rate of PBuV was 16.7% in Chinese domestic pigs in the Guangdong, Guangxi, Fujian, Jiangxi, Anhui, and Henan provinces. Additionally, the positive rate of fecal specimens was higher than that of the serum samples. Next, we sequenced nine near-complete genomes of Chinese field PBuV strains from different provinces. Homology and phylogenetic analyses indicated that Chinese PBuVs have high genetic variation (93.3-99.2%), showed higher nucleotide identity with an Austrian PBuV strain (KU867071.1), and developed into different branches within the same cluster. CONCLUSION: To our knowledge, this is the first report on PBuV in China, expanding the geographic boundaries of PBuV circulation. Our data demonstrate that PBuVs are widely distributed in the six Chinese provinces. Moreover, these Chinese PBuVs exhibit genetic variation and continuous evolution characteristics. Taken together, our findings provide a foundation for future studies on bufaviruses.