RESUMEN
Type I interferon (IFN-I) response plays a prominent role in innate immunity, which is frequently modulated during viral infection. Here, we report DNA methylation regulator UHRF1 as a potent negative regulator of IFN-I induction during alphaherpesvirus infection, whereas the viruses in turn regulates the transcriptional expression of UHRF1. Knockdown of UHRF1 in cells significantly increases interferon-ß (IFN-ß)-mediated gene transcription and viral inhibition against herpes simplex virus 1 (HSV1) and pseudorabies virus (PRV). Mechanistically, UHRF1 deficiency promotes IFN-I production by triggering dsRNA-sensing receptor RIG-I and activating IRF3 phosphorylation. Knockdown of UHRF1 in cells upregulates the accumulation of double-stranded RNA (dsRNA), including host endogenous retroviral sequence (ERV) transcripts, while the treatment of RNase III, known to specifically digest dsRNA, prevents IFN-ß induction by siUHRF1. Furthermore, the double-knockdown assay of UHRF1 and DNA methyltransferase DNMT1 suggests that siUHRF1-mediated DNA demethylation may play an important role in dsRNA accumulation and subsequently IFN induction. These findings establish the essential role of UHRF1 in IFN-I-induced antiviral immunity and reveal UHRF1 as a potential antivrial target. IMPORTANCE Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals, which rely partly on their interaction with IFN-mediated innate immune response. Using alphaherpesviruses PRV and HSV-1 as models, we identified an essential role of DNA methylation regulator UHRF1 in IFN-mediated immunity against virus replication, which unravels a novel mechanism employed by epigenetic factor to control IFN-mediated antiviral immune response and highlight UHRF1, which might be a potential target for antiviral drug development.
Asunto(s)
Herpesvirus Humano 1 , Herpesvirus Suido 1 , Interferón Tipo I , Animales , Humanos , Antivirales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Expresión Génica , Herpesvirus Humano 1/genética , Herpesvirus Suido 1/genética , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Interferón beta/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Alphaherpesvirinae , Receptores Inmunológicos/inmunologíaRESUMEN
The alphaherpesvirus pseudorabies virus (PRV) is the etiologic agent of swine Aujeszky's disease, which can cause huge economic losses to the pig industry. PRV can overcome a type I interferon (IFN)-induced antiviral state in host cells through its encoded EP0 protein. However, the exact role of EP0 in this process is poorly defined. Here, we report that EP0 transcriptionally represses IFN regulatory factor 9 (IRF9), a critical component in the IFN signaling pathway, thereby reducing the cellular levels of IRF9 and inhibiting IFN-induced gene transcription. This activity of EP0 is mediated by its C-terminal region independently of the RING domain. Moreover, compared with EP0 wild-type PRV, EP0-deficient PRV loses the ability to efficiently decrease cellular IRF9, while reintroducing the C-terminal region of EP0 back into the EP0-deficient virus restores the activity. Together, these results suggest that EP0 can transcriptionally modulate IRF9-mediated antiviral pathways through its C-terminal region, contributing to PRV innate immune evasion. IMPORTANCE Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals. Pseudorabies virus (PRV) is a swine alphaherpesvirus that threatens pig production. Using PRV as a model, we found that alphaherpesvirus can utilize its encoded early protein EP0 to inhibit the IFN-induced upregulation of antiviral proteins by reducing the basal expression levels of IRF9 through repressing its transcription. Our findings reveal a mechanism employed by alphaherpesvirus to evade the immune response and indicate that EP0 is an important viral protein in pathogenesis and a potential target for antiviral drug development.
Asunto(s)
Herpesvirus Suido 1 , Interferón Tipo I , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón , Seudorrabia , Enfermedades de los Porcinos , Animales , Antivirales/farmacología , Regulación de la Expresión Génica/inmunología , Herpesvirus Suido 1/inmunología , Herpesvirus Suido 1/metabolismo , Interacciones Microbiota-Huesped/inmunología , Interferón Tipo I/metabolismo , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Seudorrabia/inmunología , Seudorrabia/virología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
Promyelocytic leukemia nuclear bodies (PML-NBs) possess an important intrinsic antiviral activity against alphaherpesvirus infection. PML is the structural backbone of NBs, comprising different isoforms. However, the contribution of each isoform to alphaherpesvirus restriction is not well understood. Here, we report the role of PML-NBs and swine PML (sPML) isoforms in pseudorabies virus (PRV) infection in its natural host swine cells. We found that sPML-NBs exhibit an anti-PRV activity in the context of increasing the expression level of endogenous sPML. Of four sPML isoforms cloned and examined, only isoforms sPML-II and -IIa, not sPML-I and -IVa, expressed in a sPML knockout cells inhibit PRV infection. Both the unique 7b region of sPML-II and the sumoylation-dependent normal formation of PML-NBs are required. 7b possesses a transcriptional repression activity and suppresses viral gene transcription during PRV infection with the cysteine residues 589 and 599 being critically involved. We conclude that sPML-NBs inhibit PRV infection partly by repressing viral gene transcription through the 7b region of sPML-II.IMPORTANCE PML-NBs are nuclear sites that mediate the antiviral restriction of alphaherpesvirus gene expression and replication. However, the contribution of each PML isoform to this activity of PML-NBs is not well characterized. Using PRV and its natural host swine cells as a system, we have discovered that the unique C terminus of sPML isoform II is required for PML-NBs to inhibit PRV infection by directly engaging in repression of viral gene transcription. Our study not only confirms in swine cells that PML-NBs have an antiviral function but also presents a mechanism to suggest that PML-NBs inhibit viral infection in an isoform specific manner.
Asunto(s)
Herpesvirus Suido 1/genética , Cuerpos de Inclusión Intranucleares/genética , Proteína de la Leucemia Promielocítica/genética , Transcripción Genética , Proteínas Virales/genética , Animales , Línea Celular , Células Epiteliales/metabolismo , Células Epiteliales/virología , Regulación de la Expresión Génica , Células HEK293 , Herpesvirus Suido 1/metabolismo , Herpesvirus Suido 1/patogenicidad , Interacciones Huésped-Patógeno/genética , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Cuerpos de Inclusión Intranucleares/virología , Macrófagos/metabolismo , Macrófagos/virología , Proteína de la Leucemia Promielocítica/metabolismo , Dominios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Sumoilación , Porcinos , Proteínas Virales/metabolismoRESUMEN
Type I interferon response plays a prominent role against viral infection, which is frequently disrupted by viruses. Here, we report Bcl-2 associated transcription factor 1 (Bclaf1) is degraded during the alphaherpesvirus Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) infections through the viral protein US3. We further reveal that Bclaf1 functions critically in type I interferon signaling. Knockdown or knockout of Bclaf1 in cells significantly impairs interferon-α (IFNα) -mediated gene transcription and viral inhibition against US3 deficient PRV and HSV-1. Mechanistically, Bclaf1 maintains a mechanism allowing STAT1 and STAT2 to be efficiently phosphorylated in response to IFNα, and more importantly, facilitates IFN-stimulated gene factor 3 (ISGF3) binding with IFN-stimulated response elements (ISRE) for efficient gene transcription by directly interacting with ISRE and STAT2. Our studies establish the importance of Bclaf1 in IFNα-induced antiviral immunity and in the control of viral infections.
Asunto(s)
Interferones/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Virales/metabolismo , Alphaherpesvirinae/metabolismo , Alphaherpesvirinae/patogenicidad , Animales , Antivirales/farmacología , Línea Celular , China , Herpesvirus Humano 1/metabolismo , Herpesvirus Suido 1/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Interferón Tipo I/inmunología , Subunidad alfa del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Interferón-alfa/metabolismo , Interferones/inmunología , Ratones , Ratones Endogámicos BALB C , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/fisiología , Elementos de Respuesta , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Transducción de Señal/inmunología , Proteínas Supresoras de Tumor/fisiología , Proteínas Virales/genética , Virosis/genéticaRESUMEN
Alphaherpesviruses that establish persistent infections rely partly on their ability to evade host antiviral responses, notably the type I interferon (IFN) response. However, the mechanisms employed by alphaherpesviruses to avoid this response are not well understood. Pseudorabies virus (PRV) is an economically important pathogen and a useful model system for studying alphaherpesvirus biology. To identify PRV proteins that antagonize type I IFN signaling, we performed a screen by using an IFN-stimulated response element reporter in the swine cell line CRL. Unexpectedly, we identified the dUTPase UL50 as a strong inhibitor. We confirmed that UL50 has the ability to inhibit type I IFN signaling by performing ectopic expression of UL50 in cells and deletion of UL50 in PRV. Mechanistically, UL50 impeded type I IFN-induced STAT1 phosphorylation, likely by accelerating lysosomal degradation of IFN receptor 1 (IFNAR1). In addition, this UL50 activity was independent of its dUTPase activity and required amino acids 225 to 253 in the C-terminal region. The UL50 encoded by herpes simplex virus 1 (HSV-1) also possessed similar activity. Moreover, UL50-deleted PRV was more susceptible to IFN than UL50-proficient PRV. Our results suggest that in addition to its dUTPase activity, the UL50 protein of alphaherpesviruses possesses the ability to suppress type I IFN signaling by promoting lysosomal degradation of IFNAR1, thereby contributing to immune evasion. This finding reveals UL50 as a potential antiviral target.IMPORTANCE Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals. Pseudorabies virus (PRV) is a swine alphaherpesvirus that threatens pig production. Using PRV as a model, we found that this alphaherpesvirus could utilize its encoded dUTPase UL50 to induce IFNAR1 degradation and inhibit type I IFN signaling in an enzymatic activity-independent manner. Our finding reveals a mechanism employed by an alphaherpesvirus to evade the immune response and indicates that UL50 is an important viral protein in pathogenesis and is a potential target for antiviral drug development.
Asunto(s)
Herpesvirus Suido 1/enzimología , Interferón Tipo I/farmacología , Lisosomas/metabolismo , Seudorrabia/metabolismo , Pirofosfatasas/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/farmacología , Células HeLa , Herpesvirus Suido 1/genética , Humanos , Evasión Inmune , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/virología , Fosforilación , Proteolisis , Seudorrabia/tratamiento farmacológico , Seudorrabia/virología , Pirofosfatasas/genética , Receptor de Interferón alfa y beta/genética , Homología de Secuencia , Transducción de Señal , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Retroviral integrase catalyzes the integration of retroviral genome into host chromosomal DNA, which is a prerequisite of effective viral replication and infection. The human immunodeficiency virus type 1 (HIV-1) integrase has previously been reported to be regulated by the ubiquitination, but the molecular characterization of integrase ubiquitination is still unclear. In this study, we analyzed the ubiquitination of avian leukosis virus (ALV) integrase in detail. The ubiquitination assay showed that, like HIV-1, ALV integrase could also be modified by ubiquitination when expressed in 293 T and DF-1 cells. Domain mapping analysis revealed that the ubiquitination of ALV integrase might mainly occurred in the catalytic core and the N-terminal zinc-binding domains. Both lysine and non-lysine residues within integrase of ALV and HIV-1 were responsible for the ubiquitin conjugation, and the N-terminal HHCC zinc-binding motif might play an important role in mediating integrase ubiquitination. Interestingly, mass spectrometry analysis identified the Thr10 and Cys37 residues in the HHCC zinc-binding motif as the ubiquitination sites, indicating that ubiquitin may be conjugated to ALV integrase through direct interaction with the non-lysine residues. These findings revealed the detailed features of retroviral integrase ubiquitination and found a novel mechanism of ubiquitination mediated by the non-lysine residues within the N-terminal zinc-binding domain of integrase.
Asunto(s)
Virus de la Leucosis Aviar/enzimología , Integrasa de VIH/química , Integrasa de VIH/metabolismo , Integrasas/química , Integrasas/metabolismo , Proteínas de los Retroviridae/química , Proteínas de los Retroviridae/metabolismo , Retroviridae/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Virus de la Leucosis Aviar/genética , Virus de la Leucosis Aviar/fisiología , Línea Celular , Pollos , Células HEK293 , Integrasa de VIH/genética , VIH-1/enzimología , VIH-1/genética , VIH-1/fisiología , Humanos , Integrasas/genética , Lisina/química , Mutagénesis Sitio-Dirigida , Retroviridae/genética , Retroviridae/fisiología , Proteínas de los Retroviridae/genética , Ubiquitinación , Zinc/metabolismoRESUMEN
UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-ß production by PRRSV was dependent on the blocking of NF-κB signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-ß expression. Moreover, we verified that nsp4 inhibited NF-κB activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKKß. nsp4 was shown to target the NF-κB essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-ß production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-κB signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-κB-mediated IFN-ß production. We found that nsp4 interfered with the NF-κB signaling pathway through the cleavage of NEMO (a key regulator of NF-κB signaling) at the E349-S350 site, leading to the downregulation of IFN-ß production induced by poly(I·C). The data presented here may help us to better understand PRRSV pathogenesis.
Asunto(s)
Interferón beta/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Regulación hacia Abajo , Interacciones Huésped-Patógeno , Interferón beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/metabolismo , Macrófagos/virología , FN-kappa B/genética , FN-kappa B/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Transducción de Señal , Porcinos , Proteínas no Estructurales Virales/genéticaRESUMEN
OBJECTIVES: The broad host range of pseudorabies virus (PRV) and large capacity for foreign DNA make it a promising vector for the development of vaccines and agents of gene therapy. RESULTS: We show that up to 100 % viral gene disrupting efficiency was achieved by simple co-transfection of the purified PRV genomes with the clustered regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) into cells. Furthermore, CRISPR/Cas9-mediated knock-in of >4-kb-long DNA cassettes into the PRV genome at a positive rate of 50 % by a homology-independent DNA repair mechanism without constructing homology arms. This approach requires only a simple plasmid construction and is applicable to knock-in of other foreign genes. CONCLUSION: Our studies offered simple and efficient methods to manipulate PRV.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Sustitución del Gen/métodos , Genética Microbiana/métodos , Genoma Viral , Herpesvirus Suido 1/genética , Portadores de Fármacos , Vectores Genéticos , Plásmidos , Recombinación Genética , Factores de TiempoRESUMEN
The retroviral integrase plays an essential role in the integration of reverse-transcribed retroviral cDNA into the host cell genome, and serves as an important target for anti-viral therapeutics. In this study, we identified the COP9 signalosome subunit 6 (CSN6) as a novel avian leukosis virus (ALV) integrase binding protein. Co-immunoprecipitation and GST pull-down assays showed that CSN6 bound to ALV integrase likely through direct interaction of CSN6 to the catalytic core of the integrase. We further demonstrated CSN6 inhibited integrase activity in vitro; knockdown of CSN6 in DF-1 promoted ALV production. These results indicated that CSN6 may be a negative regulator of ALV replication by binding to and inhibiting integrase. Our findings provided the insight into the integrase-based host defense system and may have implications in the development of integrase-based anti-viral strategies.
Asunto(s)
Virus de la Leucosis Aviar/enzimología , Integrasas/metabolismo , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/metabolismo , Virus de la Leucosis Aviar/fisiología , Secuencia de Bases , Complejo del Señalosoma COP9 , Dominio Catalítico , Cartilla de ADN , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa , Unión Proteica , Replicación ViralRESUMEN
Cancer is the main cause of global mortality, and thus far, effective therapeutic strategies for cancer treatment are in high demand. Adoptive transfer of tumorinfiltrating lymphocytes (TILs) represents a promising avenue in immunotherapy for the management of malignancies. The clinical safety and efficacy of TILbased therapy have been established through numerous rigorous clinical trials. However, the efficacy of TIL infusion in inducing an antitumor response is limited to a subset of clinical patients with cancer. Therefore, there is an urgent need to develop innovative strategies aimed at enhancing the effectiveness of TILbased therapy. In the present review, the developmental history of TILbased therapy was systematically summarized and analyzed, while also presenting a unique perspective on enhancing the multidimensional antitumor capabilities of TILs. The insight and conclusions presented in this review may contribute to improving the efficacy of TILbased therapy and expediting its development.
Asunto(s)
Inmunoterapia Adoptiva , Linfocitos Infiltrantes de Tumor , Neoplasias , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/trasplante , Neoplasias/terapia , Neoplasias/inmunología , Inmunoterapia Adoptiva/métodos , Microambiente Tumoral/inmunologíaRESUMEN
Oncolytic viruses combined with immunotherapy offer significant potential in tumor therapy. In this study, we engineered a further attenuated pseudorabies virus (PRV) vaccine strain that incorporates a PD-L1 inhibitor and demonstrated its promise as an oncolytic virus in tumor therapy. We first showed that the naturally attenuated PRV vaccine strain Bartha can efficiently infect tumor cells from multiple species, including humans, mice, and dogs in vitro. We then evaluated the safety and anti-tumor efficacy of this vaccine strain and its different single-gene deletion mutants using the B16-F10 melanoma mouse model. The TK deletion strain emerged as the optimal vector, and we inserted a PD-L1 inhibitor (iPD-L1) into it using CRISPR/Cas9 technology. Compared with the control, the recombinant PRV (rPRV-iPD-L1) exhibited more dramatic anti-tumor effects in the B16-F10 melanoma mouse model. Our study suggests that PRV can be developed not only as an oncolytic virus but also a powerful vector for expressing foreign genes to modulate the tumor microenvironment.
Asunto(s)
Antígeno B7-H1 , Herpesvirus Suido 1 , Melanoma Experimental , Viroterapia Oncolítica , Virus Oncolíticos , Animales , Ratones , Virus Oncolíticos/genética , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/fisiología , Viroterapia Oncolítica/métodos , Melanoma Experimental/terapia , Humanos , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Línea Celular Tumoral , Ratones Endogámicos C57BL , Femenino , Perros , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Vectores Genéticos/genética , Inmunoterapia/métodos , Microambiente TumoralRESUMEN
Lung cancer is the leading cause of cancer-related morbidity and mortality in China. However, the effect of traditional cancer treatment is limited. Herein, we designed a therapeutic cancer vaccine based on the tumor-associated antigen mENO1, which can prevent lung cancer growth in vivo, and explored the underlying mechanism of Ag85B-ENO146-82 therapy. Lewis lung carcinoma (LLC) tumor-bearing immunocompetent C57BL/6 mice that received Ag85B-ENO146-82 treatment showed antitumor effect. Further, we detected CD8+ T, CD4+ T in LLC-bearing C57BL/6 mice to understand the impact of Ag85B-ENO146-82 therapy on antitumor capacity. The Ag85B-ENO146-82 therapy induced intensive infiltration of CD4+ and CD8+ T cells in tumors, increased tumor-specific IFN-γ and TNF-α secretion by CD8+ T cells and promoted macrophage polarization toward M1 phenotype. Flow cytometric analysis revealed that CD8+ T effector memory (TEM) cells and central memory (TCM) cells were upregulated. qPCR and ELISA analysis showed that the expression of IFN-γ and TNF-α were upregulated, whereas of IL1ß, IL6 and IL10 were downregulated. This study demonstrated that Ag85B-ENO146-82 vaccine augmented antitumor efficacy, which was CD8+ T cells dependent. Our findings paved the way for therapeutic tumor-associated antigen peptide vaccines to enhance anti-tumor immunotherapy for treatment of cancer.
Asunto(s)
Vacunas contra el Cáncer , Carcinoma Pulmonar de Lewis , Neoplasias Pulmonares , Animales , Ratones , Linfocitos T CD8-positivos , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/farmacología , Microambiente TumoralRESUMEN
PURPOSE OF WORK: The non-structural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV) functions as a 3C-like proteinase (3CLpro) and plays a pivotal role in gene expression and replication. We have examined the biochemical properties of PRRSV 3CLpro and identified those amino acid residues involved in its catalytic activity as a prelude to developing anti-PRRSV strategies. The 3C-like proteinase (3CLpro) of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in Escherichia coli and characterized. The optimal temperature and pH for its proteolytic activity were 8°C and 7.5, respectively. Na(+) (1000 mM) and K(+) (500 mM) were not inhibitory to its activity but Cu(2+), Zn(2+), PMSF and EDTA were significantly inhibitory. His(39), Asp(64) and Ser(118) residues were identified to form the catalytic triad of PRRSV 3CLpro by a series of site-directed mutagenesis analysis.
Asunto(s)
Aminoácidos/genética , Dominio Catalítico , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/enzimología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Frío , Cisteína Endopeptidasas/química , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0069387.].
RESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease in pigs caused by PRRS virus (PRRSV). Although PRRSV infection-induced cell apoptosis has been established, the related viral protein is still unknown. Here, we reported that PRRSV nonstructural protein 4 (nsp4) was a critical apoptosis inducer. Nsp4 could activate caspase-3, -8, and -9. Using truncated constructs without different domains in nsp4, we demonstrated that the full-length of nsp4 structure was required for its apoptosis-inducing activity. Furthermore, using site-directed mutagenesis to inactivate the 3C-like serine protease activity of nsp4, we showed that nsp4-induced apoptosis was dependent on its serine protease activity. The ability of nsp4 to induce apoptosis was significantly impaired by His39, Asp64, and Ser118 mutations, suggesting that His39, Asp64, and Ser118 were essential for nsp4 to trigger apoptosis. In conclusion, our present work showed that PRRSV nsp4 could induce apoptosis in host cells and might be partially responsible for the apoptosis induced by PRRSV infection. PRRSV 3C-like protease-mediated apoptosis represents the first report in the genus Arterivirus, family Arteriviridae.
Asunto(s)
Apoptosis/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Serina Proteasas/metabolismo , Proteínas Virales/farmacología , Aminoácidos/metabolismo , Animales , Biocatálisis/efectos de los fármacos , Células COS , Caspasas/metabolismo , Chlorocebus aethiops , Activación Enzimática/efectos de los fármacos , Células HeLa , Humanos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/patología , Macrófagos Alveolares/virología , Síndrome Respiratorio y de la Reproducción Porcina/patología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Eliminación de Secuencia , Porcinos , Transfección , Proteínas Virales/metabolismoRESUMEN
The amino acid sequence (TAVSPTTLR, 829-837aa) on the glycoprotein E2 of classical swine fever virus (CSFV) is a conserved and linear neutralizing epitope. In the present study, two peptides were constructed based the core sequence of this neutralizing epitope, the dendrimeric peptide (Th-B(4)) containing four copies of B cell epitope fused to one copy of promiscuous T helper (Th) cell epitope and the peptide Th-B containing a single copy of B cell epitope fused to one copy of Th cell epitope. The dendrimeric peptide Th-B(4) elicited high titers of neutralizing antibodies as detected in an indirect ELISA, blocking ELISA and neutralization test and induced a complete protection against CSFV C strain in rabbits. The Th-B elicited low titers of neutralizing antibodies and did not induce a protection in rabbits. These results suggest that the dendrimeric peptide Th-B(4) may be a promising marker vaccine candidate against CSFV and the multimerization is a requirement for development of a peptide vaccine.
Asunto(s)
Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales/inmunología , Peste Porcina Clásica/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Pruebas de Neutralización , Conejos , Porcinos , Vacunas de Subunidad/química , Vacunas de Subunidad/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/químicaRESUMEN
The development of cell-mediated immunity has been known extremely important in clearing porcine reproductive and respiratory syndrome virus (PRRSV) in infected pigs. However, the PRRS immunology regarding the interaction of T-cells and PRRSV proteins is poorly understood. To identify the T-cell immunodominant epitopes on the membrane (M) protein of PRRSV, a series of 31 overlapping pentadecapeptides covering the entire M protein were designed and synthesized. These peptides were screened by ELIspot analysis for their capabilities to elicit interferon-gamma (IFN-γ) responses in the peripheral blood mononuclear cells (PBMCs), which were collected from pigs immunized with attenuated PRRSV HuN4-F112 strain and challenged with highly pathogenic HuN4 strain. After three rounds of screening, 4 peptides (M3, M6, M8 and M12) were shown to elicit high expression of IFN-γ. The stimulation of high IFN-γ transcription in PBMCs by these 4 peptides was further confirmed in real-time PCR. The sequence alignment revealed that the epitope represented by peptide M6 was fully conserved in all of examined 42 North American genotype II PRRSV isolates and the epitopes represented by peptides M3, M8 and M12 showed 2-4 amino acid replacements. The finding of 4 T-cell immunodominant epitopes in the M protein of PRRSV will be beneficial to the understanding of the development of cell-mediated immunity.
Asunto(s)
Epítopos de Linfocito T/inmunología , Epítopos Inmunodominantes/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Ensayo de Immunospot Ligado a Enzimas , Mapeo Epitopo , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , PorcinosRESUMEN
Since April 2006, swine herds have experienced the outbreaks of a highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China. To explore the possible mechanism of the emergence of the highly pathogenic PRRS and more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the ORF5 gene sequences of 159 representative PRRSV isolates in 16 provinces from 2006 to 2008. Sequence and phylogenetic analyses showed that all these 159 isolates belonged to the North American genotype and were further divided into six subgenotypes; 140 of 159 isolates were closely related to the highly pathogenic PRRSV with 98.5-100% nucleotide and 98.3-100% amino acid sequence identities and belonged to Subgenotype I; and 3, 8, 4, 3, 1 of 159 isolates were part of Subgenotypes II-VI, respectively. Amino acid analysis of the GP5 protein revealed that all the isolates in Subgenotypes I-III were found to be highly variable in the primary neutralizing epitope; most of the isolates in Subgenotypes I and IV had more glycosylation sites than those in Subgenotypes II, III, V and VI; and 1, 5, and 9 unique amino acid mutations were observed in Subgenotypes I, IV and VI, respectively. In conclusion, our study provides the evidence of coexistence of six different subgenotype isolates in pigs in China from 2006 to 2008, and emphasizes the importance of reinforcing PRRSV surveillance, especially after the emergence of highly pathogenic PRRS in China.