RESUMEN
IMPORTANCE: The type-I interferon (IFN-I) signaling pathway is the first line of antiviral innate immunity. It must be precisely regulated against virus-induced damage. The tightly regulated mechanisms of action of host genes in the antiviral innate immune signaling pathway are still worth studying. Here, we report a novel role of DLG1 in positively regulating the IκB kinase epsilon (IKKε)-mediated IFN-I signaling response against negative-stranded RNA virus replication, whereas the RNA virus inhibits the expression of DLG1 for immune escape. Importantly, the E3 ligase March2 interacts with and promotes K27-linked polyubiquitination of IKKε, and p62 is a cargo receptor that recognizes ubiquitinated IKKε for eventual autophagic degradation. Together, the current findings elucidate the role of DLG1 in the antiviral IFN-I signaling pathway and viral infection repression.
Asunto(s)
Autofagia , Homólogo 1 de la Proteína Discs Large , Quinasa I-kappa B , Inmunidad Innata , Virus ARN de Sentido Negativo , Proteína Sequestosoma-1 , Virosis , Humanos , Homólogo 1 de la Proteína Discs Large/metabolismo , Quinasa I-kappa B/metabolismo , Inmunidad Innata/inmunología , Virus ARN de Sentido Negativo/crecimiento & desarrollo , Virus ARN de Sentido Negativo/inmunología , Poliubiquitina/metabolismo , Proteína Sequestosoma-1/antagonistas & inhibidores , Transducción de Señal , Virosis/inmunología , Animales , Línea CelularRESUMEN
DNA damage-inducible transcript 3 (DDIT3) plays important roles in endoplasmic reticulum (ER) stress-induced apoptosis and autophagy, but its role in innate immunity is not clear. Here, we report that DDIT3 inhibits the antiviral immune response during bovine viral diarrhea virus (BVDV) infection by targeting mitochondrial antiviral signaling (MAVS) in Madin-Darby bovine kidney (MDBK) cells and in mice. BVDV infection induced high DDIT3 mRNA and protein expression. DDIT3 overexpression inhibited type I interferon (IFN-I) and IFN-stimulated gene production, thereby promoting BVDV replication, while DDIT3 knockdown promoted the antiviral innate immune response to suppress viral replication. DDIT3 promoted NF-κB-dependent ovarian tumor (OTU) deubiquitinase 1 (OTUD1) expression. Furthermore, OTUD1 induced upregulation of the E3 ubiquitin ligase Smurf1 by deubiquitinating Smurf1, and Smurf1 degraded MAVS in MDBK cells in a ubiquitination-dependent manner, ultimately inhibiting IFN-I production. Moreover, knocking out DDIT3 promoted the antiviral innate immune response to reduce BVDV replication and pathological changes in mice. These findings provide direct insights into the molecular mechanisms by which DDIT3 inhibits IFN-I production by regulating MAVS degradation.IMPORTANCE Extensive studies have demonstrated roles of DDIT3 in apoptosis and autophagy during viral infection. However, the role of DDIT3 in innate immunity remains largely unknown. Here, we show that DDIT3 is positively regulated in bovine viral diarrhea virus (BVDV)-infected Madin-Darby bovine kidney (MDBK) cells and could significantly enhance BVDV replication. Importantly, DDIT3 induced OTU deubiquitinase 1 (OTUD1) expression by activating the NF-κB signaling pathway, thus increasing intracellular Smurf1 protein levels to degrade MAVS and inhibit IFN-I production during BVDV infection. Together, these results indicate that DDIT3 plays critical roles in host innate immunity repression and viral infection facilitation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Virus de la Diarrea Viral Bovina Tipo 1/fisiología , Inmunidad Innata , Factor de Transcripción CHOP/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Replicación Viral , Animales , Antivirales/antagonistas & inhibidores , Antivirales/inmunología , Bovinos , Virus de la Diarrea Viral Bovina Tipo 1/patogenicidad , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Ratones , FN-kappa B/metabolismo , Transducción de Señal , Factor de Transcripción CHOP/deficiencia , Factor de Transcripción CHOP/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/genética , UbiquitinaciónRESUMEN
BACKGROUND: As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS: A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS: The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS: Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.
Asunto(s)
Virus de la Diarrea Viral Bovina Tipo 1/aislamiento & purificación , Genotipo , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Regiones no Traducidas 5'/genética , Aerosoles , Microbiología del Aire , Animales , Virus de la Diarrea Viral Bovina Tipo 1/clasificación , Virus de la Diarrea Viral Bovina Tipo 1/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y EspecificidadRESUMEN
Bovine viral diarrhea virus (BVDV) is a worldwide spreading pestivirus affecting cattle and other ruminants; however, there have been few reports on epidemiologic investigation of BVDV in eastern China. In this study, bulk tank milk from 36 herds of dairy cattle in eastern China was submitted to serological investigations, 77.8% of herds was BVDV antibody positive. Individual animal status in two herds was further investigated collecting blood samples, the positive ratio was 49.74% and 24.64%, and the average positive ratio of calves, heifers, and lactating cows was 15.94%, 40.16%, and 41.7%, respectively. Moreover, clinical survey was carried out among 8170 dairy cattle from 36 herds, for diarrhea syndrome, respiratory problems and reproductive failure, and pathogens of all clinical cattle were further investigated. The results showed that BVDV was one of the main pathogen, which infected animals combining with various other viruses. Then, nine BVDV strains were isolated; phylogenetic analysis showed that BVDV subtypes currently circulating in eastern China were BVDV 1a and BVDV 1c. In addition, out of 377 cows tested, the 1.86% detected positive to the BVDV antigen. This study provided the foundation of further study on vaccination and control strategies of BVDV in eastern China.
Asunto(s)
Anticuerpos Antivirales/sangre , Diarrea Mucosa Bovina Viral/epidemiología , Virus de la Diarrea Viral Bovina Tipo 1/aislamiento & purificación , Animales , Diarrea Mucosa Bovina Viral/prevención & control , Diarrea Mucosa Bovina Viral/virología , Bovinos , China/epidemiología , Industria Lechera , Virus de la Diarrea Viral Bovina Tipo 1/genética , Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Femenino , Leche/virología , Filogenia , Prevalencia , Vacunación/veterinariaRESUMEN
BACKGROUND: Bovine ephemeral fever virus (BEFV), the causative agent of bovine ephemeral fever, is an economically important pathogen of cattle and water buffalo. MicroRNAs (miRNAs) are endogenous 21-23 nt small non-coding RNA molecules that binding to a multiple of target mRNAs and functioning in the regulation of viral replication including the miRNA-mediated antiviral defense. However, the reciprocal interaction between bovine ephemeral fever virus replication and host miRNAs still remain poorly understood. The aim of our study herein was to investigate the exact function of miR-3470b and its molecular mechanisms during BEFV infection. RESULTS: In this study, we found a set of microRNAs induced by BEFV infection using small RNA deep sequencing, and further identified BEFV infection could significantly up-regulate the miR-3470b expression in Baby Hamster Syrian Kidney cells (BHK-21) after 24 h and 48 h post-infection (pi) compared to normal BHK-21 cells without BEFV infection. Additionally, the target association between miR-3470b and mitochondrial antiviral signaling protein (MAVS) was predicted by target gene prediction tools and further validated using a dual-luciferase reporter assay, and the expression of MAVS mRNA and protein levels was negatively associated with miR-3470b levels. Furthermore, the miR-3470b mimic transfection significantly contributed to increase the BEFV N mRNA, G protein level and viral titer, respectively, whereas the miR-3470b inhibitor had the opposite effect on BEFV replication. Moreover, the overexpression of MAVS or silencing of miR-3470b by its inhibitors suppressed BEFV replication, and knockdown of MAVS by small interfering RNA also promoted the replication of BEFV. CONCLUSIONS: Our findings is the first to reveal that miR-3470b as a novel host factor regulates BEFV replication via directly targeting the MAVS gene in BHK-21 cells and may provide a potential strategy for developing effective antiviral therapy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Virus de la Fiebre Efímera Bovina/fisiología , Fiebre Efímera/inmunología , Fiebre Efímera/virología , Riñón/inmunología , MicroARNs/genética , Replicación Viral , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Bovinos , Cricetinae , Fiebre Efímera/genética , Virus de la Fiebre Efímera Bovina/genética , Interacciones Huésped-Patógeno , Riñón/virología , Mesocricetus , MicroARNs/inmunología , ConejosRESUMEN
Bovine respirovirus 3 also known as Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory agents of both young and adult cattle. Rapid diagnosis could contribute greatly in containing epidemics and thus avoid economic losses. However, the lack of robust isothermal visual method poses difficulty. In this study, a novel isothermal assay for detecting BPIV3 was established. The method includes a lateral flow dipstick (LFD) assay combined with reverse transcription recombinase polymerase amplification (RT-RPA). First, the analytical sensitivity and specificity of BPIV3 LFD RT-RPA were tested. The LFD RT-RPA assay has a detection limit of up to 100 copies per reaction in 30â¯minâ¯at 38⯰C. Then the performance of LFD RT-RPA was evaluated using 95 clinical samples. Compared to qPCR, the LFD RT-RPA assay showed a clinical sensitivity of 94.74%, a clinical specificity of 96.05% and 0.8734 kappa coefficient. These results have demonstrated the efficiency and effectiveness of the method to be developed into a point of care protocol for the diagnosis of BPIV3.
Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Recombinasas/metabolismo , Respirovirus/aislamiento & purificación , Transcripción Reversa/genética , Reología , Animales , Bovinos , Valor Predictivo de las Pruebas , Respirovirus/genética , Sensibilidad y EspecificidadRESUMEN
Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38⯰C for 20â¯min and LFD incubation time within 5â¯min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak.
Asunto(s)
Bioensayo/métodos , Virus de la Fiebre Efímera Bovina/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Recombinasas/metabolismo , Animales , Bovinos , Sensibilidad y Especificidad , Temperatura , Factores de TiempoRESUMEN
BACKGROUND: The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G1 protein with high-affinity and inhibit BEFV replication. METHODS: The purified BEFV G1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G1 was assayed by ELISA. Then the roles of specific G1-binding peptides in the context of BEFV infection were analyzed. RESULTS: The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. CONCLUSION: Two antiviral peptide ligands binding to bovine ephemeral fever virus G1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.
Asunto(s)
Virus de la Fiebre Efímera Bovina/fisiología , Glicoproteínas/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales , Bacteriófagos , Bovinos , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Fiebre Efímera/metabolismo , Fiebre Efímera/virología , Virus de la Fiebre Efímera Bovina/genética , Glicoproteínas/genética , Biblioteca de Péptidos , Péptidos/genética , Unión ProteicaRESUMEN
BACKGROUND: Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most highly infectious diseases in livestock, and leads to huge economic losses. Early diagnosis and rapid differentiation of FMDV serotype is therefore integral to the prevention and control of FMD. In this study, a series of serotype-specific reverse transcription recombinase polymerase amplification assays combined with lateral flow dipstick (RPA-LFD) were establish to differentiate FMDV serotypes A, O or Asia 1, respectively. RESULTS: The serotype-specific primers and probes of RPA-LFD were designed to target conserved regions of the FMDV VP1 gene sequence, and three primer and probe sets of serotype-specific RPA-LFD were selected for amplification of FMDV serotypes A, O or Asia 1, respectively. Following incubation at 38 °C for 20 min, the RPA amplification products could be visualized by LFD. Analytical sensitivity of the RPA assay was then determined with ten-fold serial dilutions of RNA of VP1 gene and the recombinant vector respectively containing VP1 gene from FMDV serotypes A, O or Asia1, the detection limits of these assays were 3 copies of plasmid DNA or 50 copies of viral RNA per reaction. Moreover, the specificity of the assay was assessed, and there was no cross reactions with other viruses leading to bovine vesicular lesions. Furthermore, 126 clinical samples were respectively detected with RPA-LFD and real-time PCR (rPCR), there was 98.41% concordance between the two assays, and two samples were positive by RPA-LFD but negative in rPCR, these were confirmed as FMDV-positive through viral isolation in BHK-21 cells. It showed that RPA-LFD assay was more sensitive than the rPCR method in this study. CONCLUSION: The development of serotype-specific RPA-LFD assay provides a rapid, sensitive, and specific method for differentiation of FMDV serotype A, O or Asia1, respectively. It is possible that the serotype-specific RPA-LFD assay may be used as a integral protocol for field detection of FMDV.
Asunto(s)
Virus de la Fiebre Aftosa , Fiebre Aftosa/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Serotipificación/veterinaria , Animales , Bovinos/virología , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/virología , Virus de la Fiebre Aftosa/clasificación , Virus de la Fiebre Aftosa/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/veterinaria , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Sensibilidad y Especificidad , Serotipificación/métodosRESUMEN
BACKGROUND: Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recombinase polymerase amplification (RPA) technique has become a promising isothermal DNA amplify assay for use in rapid and low-resource diagnostics. RESULTS: Here, a method for specific detection of M. bovis DNA was established, which was RPA combined with lateral flow dipstick (LFD). First, the analytical specificity and sensitivity of the RPA primer and LF-probe sets were evaluated. The assay successfully detected M. bovis DNA in 30 min at 39 °C, with detection limit of 20 copies per reaction, which it was compared the real-time quantitative PCR (qPCR) assay. This method was specific because it did not detect a selection of other bacterial pathogens in cattle. Both qPCR and RPA-LFD assays were used to detect M. bovis 442 field samples from 42 different dairy farms in Shandong Province of China, also the established RPA-LFD assay obtained 99.00% sensitivity, 95.61% specificity, and 0.902 kappa coefficient compared with the qPCR. CONCLUSIONS: To the author's knowledge, this is the first report using an RPA-FLD assay to visualise and detect M. bovis. Comparative analysis with qPCR indicates the potential of this assay for rapid diagnosis of bovine mycoplasmosis in resource limited settings.
Asunto(s)
Enfermedades de los Bovinos/diagnóstico , Técnicas de Diagnóstico Molecular/veterinaria , Infecciones por Mycoplasma/veterinaria , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Animales , Bovinos , China , ADN Polimerasa Dirigida por ADN/metabolismo , Infecciones por Mycoplasma/diagnóstico , Mycoplasma bovis/genética , Técnicas de Amplificación de Ácido Nucleico/normas , Recombinasas/metabolismo , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV. METHODS: Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens. RESULTS: RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%. CONCLUSION: IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.
Asunto(s)
Rinotraqueítis Infecciosa Bovina/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Animales , Bovinos , ADN Viral/genética , Herpesvirus Bovino 1/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The G protein-coupled receptor ADGRE5 (CD97) binds to various metabolites that play crucial regulatory roles in metabolism. However, its function in the antiviral innate immune response remains to be determined. In this study, we report that CD97 inhibits virus-induced type-I interferon (IFN-I) release and enhances RNA virus replication in cells and mice. CD97 was identified as a new negative regulator of the innate immune receptor RIG-I, and RIG-1 degradation led to the suppression of the IFN-I signaling pathway. Furthermore, overexpression of CD97 promoted the ubiquitination of RIG-I, resulting in its degradation, but did not impact its mRNA expression. Mechanistically, CD97 upregulates RNF125 expression to induce RNF125-mediated RIG-I degradation via K48-linked ubiquitination at Lys181 after RNA virus infection. Most importantly, CD97-deficient mice are more resistant than wild-type mice to RNA virus infection. We also found that sanguinarine-mediated inhibition of CD97 effectively blocks VSV and SARS-CoV-2 replication. These findings elucidate a previously unknown mechanism through which CD97 negatively regulates RIG-I in the antiviral innate immune response and provide a molecular basis for the development of new therapeutic strategies and the design of targeted antiviral agents.
Asunto(s)
Infecciones por Virus ARN , Virus ARN , Animales , Ratones , Antivirales/farmacología , Proteína 58 DEAD Box/metabolismo , Inmunidad Innata , Receptores Acoplados a Proteínas G/metabolismo , Infecciones por Virus ARN/genética , Virus ARN/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is closely related to various cellular aspects associated with autophagy. However, how SARS-CoV-2 mediates the subversion of the macroautophagy/autophagy pathway remains largely unclear. In this study, we demonstrate that overexpression of the SARS-CoV-2 ORF7a protein activates LC3-II and leads to the accumulation of autophagosomes in multiple cell lines, while knockdown of the viral ORF7a gene via shRNAs targeting ORF7a sgRNA during SARS-CoV-2 infection decreased autophagy levels. Mechanistically, the ORF7a protein initiates autophagy via the AKT-MTOR-ULK1-mediated pathway, but ORF7a limits the progression of autophagic flux by activating CASP3 (caspase 3) to cleave the SNAP29 protein at aspartic acid residue 30 (D30), ultimately impairing complete autophagy. Importantly, SARS-CoV-2 infection-induced accumulated autophagosomes promote progeny virus production, whereby ORF7a downregulates SNAP29, ultimately resulting in failure of autophagosome fusion with lysosomes to promote viral replication. Taken together, our study reveals a mechanism by which SARS-CoV-2 utilizes the autophagic machinery to facilitate its own propagation via ORF7a.Abbreviations: 3-MA: 3-methyladenine; ACE2: angiotensin converting enzyme 2; ACTB/ß-actin: actin beta; ATG7: autophagy related 7; Baf A1: bafilomycin A1; BECN1: beclin 1; CASP3: caspase 3; COVID-19: coronavirus disease 2019; GFP: green fluorescent protein; hpi: hour post-infection; hpt: hour post-transfection; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERS: Middle East respiratory syndrome; MTOR: mechanistic target of rapamycin kinase; ORF: open reading frame; PARP: poly(ADP-ribose) polymerase; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; shRNAs: short hairpin RNAs; siRNA: small interfering RNA; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TCID50: tissue culture infectious dose; TEM: transmission electron microscopy; TUBB, tubulin, beta; ULK1: unc-51 like autophagy activating kinase 1.
Asunto(s)
Autofagia , COVID-19 , Humanos , Autofagosomas/metabolismo , Autofagia/genética , Caspasa 3/metabolismo , Lisosomas/metabolismo , Macroautofagia , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE , SARS-CoV-2 , Serina-Treonina Quinasas TOR/metabolismo , Replicación ViralRESUMEN
OBJECTIVE: To identify the interaction between influenza A virus PB1-F2 and human modulator of apoptosis 1 (MOAP-1). METHODS: The recombinant plasmid pACT2-MOAP-1 was constructed and co-transformed into yeast AH109 with pGBKT7-PB1-F2. The growth of the co-transformants on quadruple dropout medium and beta-galactosidase activity of the reporter gene were tested. We further confirmed the interaction of cellular protein MOAP-1 and PB1-F2 by glutathione S-transferase (GST) pull-down and co-immunoprecipitation (Co-IP) assays. In addition, we investigated the effect of PB1-F2 on MOAP-1 protein level by Western blot. RESULTS: The results of yeast two-hybrid assay showed that MOAP-1 specifically interacted with PB1-F2 in yeast cells. Furthermore, the binding of MOAP-1 with PB1-F2 was demonstrated by glutathione S-transferase pull-down and Co-IP assays. PB1-F2 could upregulate exogenous MOAP-1 protein level. CONCLUSION: These results suggested that influenza virus PB1-F2 interacted with MOAP-1 and it might be involved in the regulation of cell growth and apoptosis via association with MOAP-1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Proteínas Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/genética , Unión Proteica , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genéticaRESUMEN
The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused severe morbidity and mortality in humans. It is urgent to understand the function of viral genes. However, the function of open reading frame 10 (ORF10), which is uniquely expressed by SARS-CoV-2, remains unclear. In this study, we showed that overexpression of ORF10 markedly suppressed the expression of type I interferon (IFN-I) genes and IFN-stimulated genes. Then, mitochondrial antiviral signaling protein (MAVS) was identified as the target via which ORF10 suppresses the IFN-I signaling pathway, and MAVS was found to be degraded through the ORF10-induced autophagy pathway. Furthermore, overexpression of ORF10 promoted the accumulation of LC3 in mitochondria and induced mitophagy. Mechanistically, ORF10 was translocated to mitochondria by interacting with the mitophagy receptor Nip3-like protein X (NIX) and induced mitophagy through its interaction with both NIX and LC3B. Moreover, knockdown of NIX expression blocked mitophagy activation, MAVS degradation, and IFN-I signaling pathway inhibition by ORF10. Consistent with our observations, in the context of SARS-CoV-2 infection, ORF10 inhibited MAVS expression and facilitated viral replication. In brief, our results reveal a novel mechanism by which SARS-CoV-2 inhibits the innate immune response; that is, ORF10 induces mitophagy-mediated MAVS degradation by binding to NIX.
Asunto(s)
COVID-19/genética , COVID-19/virología , Inmunidad Innata/inmunología , Sistemas de Lectura Abierta , SARS-CoV-2/genética , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antivirales/metabolismo , Autofagia/inmunología , Silenciador del Gen , Células HEK293 , Células HeLa , Humanos , Interferón Tipo I/metabolismo , Mitocondrias/metabolismo , Mitofagia , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Receptors for activated C kinase 1 (RACK1) could competitively combine with mitochondrial antiviral signaling protein (MAVS) to inhibit the type I interferon (IFN) signaling pathway during viral infection in vitro. However, whether RACK1 can degrade MAVS to enhance viral replication is still unknown. In this study, we found that bovine epidemic fever virus (BEFV) infection triggered the expression of RACK1. Overexpression of RACK1 promoted BEFV replication, while knockdown of RACK1 inhibited the replication of BEFV. Further research showed that RACK1 inhibited the type I IFN signaling pathway during BEFV infection by degrading MAVS, and RACK1 degraded MAVS via the ubiquitin-proteasome system. Mechanistically, RACK1 up-regulated the expression of E3 ubiquitin ligase STIP1 homology and U-box containing protein 1 (STUB1), thereby promoting the ubiquitination and degradation of MAVS. In addition, RACK1 degraded MAVS by enhancing the interaction between STUB1 and MAVS but not via its interaction with STUB1. Overall, our study reveals a novel mechanism by which RACK1 inhibits the type I IFN signaling pathway to BEFV infection through degradation of MAVS, thereby promoting viral infection. These findings provide a new perspective for the MAVS degradation regulated by RACK1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Virus de la Fiebre Efímera Bovina/fisiología , Inmunidad Innata , Receptores de Cinasa C Activada/genética , Ubiquitina-Proteína Ligasas/genética , Regulación hacia Arriba , Replicación Viral/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Bovinos , Línea Celular , Cricetinae , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Células HEK293 , Humanos , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/inmunología , Transducción de Señal/inmunologíaRESUMEN
MicroRNAs (miRNAs), as a kind of small noncoding RNAs, have been proved to play a regulatory role in virus infection. However, the role and mechanism of cellular miRNAs in bovine transient fever virus (BEFV) infection are largely unknown. In the present study, we found that bta-miR-101 was significantly up-regulated in the Madin-Darby Bovine Kidney (MDBK) cells upon BEFV infection. Notably, bta-miR-101 mimic dramatically inhibited BEFV replication, while bta-miR-101 inhibitor facilitated BEFV replication, suggesting that bta-miR-101 acted as an anti-viral host factor restraining BEFV replication. Subsequently, NF-κB repressing factor (NKRF) was identified as a target gene of bta-miR-101 by dual luciferase reporter assay, and bta-miR-101 mimic significantly down-regulated expression of NKRF, while bta-miR-101 inhibitor up-regulated its expression, respectively. Furthermore, NKRF could induce apoptosis, and favored the replication of BEFV. Finally, bta-miR-101 inhibited BEFV-induced apoptosis via targeting NKRF to suppress virus replication. In general, our study provides a novel mechanism for bta-miR-101 to exert its antiviral function, which provides a theoretical basis for the development of antiviral strategy.
Asunto(s)
Virus de la Fiebre Efímera Bovina/genética , Células Epiteliales/virología , Interacciones Huésped-Patógeno/genética , MicroARNs/genética , Proteínas Represoras/genética , Replicación Viral/genética , Animales , Bovinos , Línea Celular , Regulación hacia Abajo , Células HEK293 , Humanos , Riñón/citología , Regulación hacia ArribaRESUMEN
Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus and causes bovine ephemeral fever of cattle and water buffalo in worldwide. Previous studies have demonstrated that infection with BEFV leads to induction of host cellular apoptosis. However, the role of apoptosis in viral replication and the interaction between viral genes and host genes involved in the process of BEFV-induced apoptosis remains unclear. Herein we investigated the interaction between viral non-structural protein α3 and cellular heterogeneous nuclear ribonucleoprotein K (hnRNP K) in the BEFV-induced apoptosis and its role in virus replication. Overexpression of α3 gene activated caspase 3 and consequently cleaved PARP, ultimately lead to apoptosis. Moreover, virus titer of BHK-21 cells infected with BEFV and then treated respectively by the pan-caspase inhibitor (Z-VAD-FMK) and apoptosis inducer (CCCP) was determined, the results showed that apoptosis promoted viral replication. In addition, knockdown of hnRNP K gene promoted BEFV replication, whereas overexpression of hnRNP K gene had the opposite effects. More importantly, overexpression of hnRNP K inhibited virus-induced apoptosis. Subsequently, it was found that hnRNP K suppressed BEFV replication via degrading viral α3 gene and further inhibited apoptosis induced by α3 gene. Finally, the expression of hnRNP K protein was significantly down-regulated upon BEFV infection, and degradation of hnRNP K protein in BHK-21 cells infected with BEFV was mediated by viral activation of caspase 3. Taken together, these results suggest that apoptosis takes a pivotal role in BEFV replication, and interaction between viral α3 gene and host hnRNP K gene in BEFV-induced apoptosis facilitates BEFV replication.
Asunto(s)
Apoptosis/genética , Virus de la Fiebre Efímera Bovina/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Interacciones Microbiota-Huesped/genética , Proteínas no Estructurales Virales/genética , Replicación Viral , Animales , Bovinos , Línea Celular , Cricetinae , Regulación hacia Abajo , Virus de la Fiebre Efímera Bovina/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del GenRESUMEN
Bovine ephemeral fever virus (BEFV) causes bovine ephemeral fever, which can produce considerable economic damage to the cattle industry. However, there is limited experimental evidence regarding the underlying mechanisms of BEFV. Annexin A2 (AnxA2) is a calcium and lipid-conjugated protein that binds phospholipids and the cytoskeleton in a Ca2+-dependent manner, and it participates in various cellular functions, including vesicular trafficking, organization of membrane domains, and virus proliferation. The role of the AnxA2 gene during virus infection has not yet been reported. In this study, we observed that AnxA2 gene expression was up-regulated in BHK-21 cells infected with the virus. Additionally, overexpression of the AnxA2 gene promoted the release of mature virus particles, whereas BEFV replication was remarkably inhibited after reducing AnxA2 gene expression by using the small interfering RNA (siRNA). For viral proteins, overexpression of the Matrix (M) gene promotes the release of mature virus particles. Moreover, the AnxA2 protein interaction with the M protein of BEFV was confirmed by GST pull-down and co-immunoprecipitation assays. Experimental results indicate that the C-terminal domain (268-334 aa) of AxnA2 contributes to this interaction. An additional mechanistic study showed that AnxA2 protein interacts with M protein and mediates the localization of the M protein at the plasma membrane. Furthermore, the absence of the AnxA2-V domain could attenuate the effect of AnxA2 on BEFV replication. These findings can contribute to elucidating the regulation of BEFV replication and may have implications for antiviral strategy development.
Asunto(s)
Anexina A2/metabolismo , Virus de la Fiebre Efímera Bovina/fisiología , Fiebre Efímera/virología , Proteínas de la Matriz Viral/metabolismo , Replicación Viral , Animales , Bovinos , Regulación hacia ArribaRESUMEN
Bovine herpesvirus 1 (BHV-1) is an economically important pathogen of cattle and has led to significant consequences on the cattle industry worldwide. MicroRNAs (miRNAs) are a class of regulators that play critical roles in virus and host interaction. However, the roles of host miRNAs in BHV-1 infection remain largely unclear. In this study, a set of differentially expressed miRNAs by small RNA deep sequencing were analyzed in the Madin-Darby Bovine Kidney Cells (MDBK) infected with BHV-1 after 12 h, 24 h and 48 h post-infection compared to mock infection, and it was confirmed that bta-miR-2361 was significantly down-regulated. Moreover, bta-miR-2361 mimics transfection could inhibit BHV-1 replication. Combined with up-regulated genes from BHV-1-infected MDBK cells by deep RNA-sequencing and predicted by bioinformatics tools, early growth response 1 (EGR1) was putative target of bta-miR-2361. Furthermore, EGR1 was up-regulated during BHV-1 infection, and overexpression of EGR1 promoted BHV-1 replication whereas knockdown of EGR1 had the opposite effects. Subsequently, the target association between bta-miR-2361 and 3'UTR of EGR1 was further validated using a dual-luciferase reporter assay. In addition, overexpression of bta-miR-2361 resulted in decreased EGR1 mRNA and protein levels. Further mechanistic study showed that EGR1 stimulated BHV-1 UL46 promoter activity, but overexpression of bta-miR-2361 suppressed the production of UL46 gene. Collectively, this is the first study to reveal that bta-miR-2361 as a novel host factor regulates BHV-1 replication via directly targeting the EGR1 gene, which is a transcription factor that regulates viral UL46 gene of BHV-1. These results provide further insight into the study of BHV-1 pathogenesis.