RESUMEN
African swine fever virus (ASFV) infection is characterized by a progressive decrease in cellular protein synthesis with a concomitant increase in viral protein synthesis, though the mechanism by which the virus achieves this is still unknown. Decrease of cellular mRNA is observed during ASFV infection, suggesting that inhibition of cellular proteins is due to an active mRNA degradation process. ASFV carries a gene (Ba71V D250R/Malawi g5R) that encodes a decapping protein (ASFV-DP) that has a Nudix hydrolase motif and decapping activity in vitro Here, we show that ASFV-DP was expressed from early times and accumulated throughout the infection with a subcellular localization typical of the endoplasmic reticulum, colocalizing with the cap structure and interacting with the ribosomal protein L23a. ASFV-DP was capable of interaction with poly(A) RNA in cultured cells, primarily mediated by the N-terminal region of the protein. ASFV-DP also interacted with viral and cellular RNAs in the context of infection, and its overexpression in infected cells resulted in decreased levels of both types of transcripts. This study points to ASFV-DP as a viral decapping enzyme involved in both the degradation of cellular mRNA and the regulation of viral transcripts.IMPORTANCE Virulent ASFV strains cause a highly infectious and lethal disease in domestic pigs for which there is no vaccine. Since 2007, an outbreak in the Caucasus region has spread to Russia, jeopardizing the European pig population and making it essential to deepen knowledge about the virus. Here, we demonstrate that ASFV-DP is a novel RNA-binding protein implicated in the regulation of mRNA metabolism during infection, making it a good target for vaccine development.
Asunto(s)
Virus de la Fiebre Porcina Africana/enzimología , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , ARN Mensajero/metabolismo , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Chlorocebus aethiops , Eliminación de Gen , Interacciones Huésped-Patógeno , Unión Proteica , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Proteínas Ribosómicas/metabolismo , Sus scrofa , Células Vero , Proteínas Virales/genética , Hidrolasas NudixRESUMEN
The African swine fever virus (ASFV) protein pE248R, encoded by the gene E248R, is a late structural component of the virus particle. The protein contains intramolecular disulfide bonds and has been previously identified as a substrate of the ASFV-encoded redox system. Its amino acid sequence contains a putative myristoylation site and a hydrophobic transmembrane region near its carboxy terminus. We show here that the protein pE248R is myristoylated during infection and associates with the membrane fraction in infected cells, behaving as an integral membrane protein. Furthermore, the protein localizes at the inner envelope of the virus particles in the cytoplasmic factories. The function of the protein pE248R in ASFV replication was investigated by using a recombinant virus that inducibly expresses the gene E248R. Under repressive conditions, the ASFV polyproteins pp220 and pp62 are normally processed and virus particles with morphology indistinguishable from that of those produced in a wild-type infection or under permissive conditions are generated. Moreover, the mutant virus particles can exit the cell as does the parental virus. However, the infectivity of the pE248R-deficient virions was reduced at least 100-fold. An investigation of the defect of the mutant virus indicated that neither virus binding nor internalization was affected by the absence of the protein pE248R, but a cytopathic effect was not induced and early and late gene expression was impaired, indicating that the protein is required for some early postentry event.
Asunto(s)
Virus de la Fiebre Porcina Africana/fisiología , Proteínas de la Membrana/fisiología , Proteínas Estructurales Virales/fisiología , Internalización del Virus , Replicación Viral , Animales , Membrana Celular/química , Efecto Citopatogénico Viral , Proteínas de la Membrana/deficiencia , Proteínas Estructurales Virales/deficienciaRESUMEN
African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Virus de la Fiebre Porcina Africana/patogenicidad , Proteínas Virales/metabolismo , Virus de la Fiebre Porcina Africana/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Macrófagos/virología , Unión Proteica , Porcinos , Proteínas Virales/químicaRESUMEN
African swine fever virus (ASFV) is able to inhibit TNF-alpha-induced gene expression through the synthesis of A238L protein. This was shown by the use of deletion mutants lacking the A238L gene from the Vero cell-adapted Ba71V ASFV strain and from the virulent isolate E70. To further analyze the molecular mechanism by which the viral gene controls TNF-alpha, we have used Jurkat cells stably transfected with the viral gene to identify the TNF-alpha regulatory elements involved in the induction of the gene after stimulation with PMA and calcium ionophore. We have thus identified the cAMP-responsive element and kappa3 sites on the TNF-alpha promoter as the responsible of the gene activation, and demonstrate that A238L inhibits TNF-alpha expression through these DNA binding sites. This inhibition was partially reverted by overexpression of the transcriptional factors NF-AT, NF-kappaB, and c-Jun. Furthermore, we present evidence that A238L inhibits the activation of TNF-alpha by modulating NF-kappaB, NF-AT, and c-Jun trans activation through a mechanism that involves CREB binding protein/p300 function, because overexpression of these transcriptional coactivators recovers TNF-alpha promoter activity. In addition, we show that A238L is a nuclear protein that binds to the cyclic AMP-responsive element/kappa3 complex, thus displacing the CREB binding protein/p300 coactivators. Taken together, these results establish a novel mechanism in the control of TNF-alpha gene expression by a viral protein that could represent an efficient strategy used by ASFV to evade the innate immune response.
Asunto(s)
Regulación Viral de la Expresión Génica , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/genética , Proteínas Virales/metabolismo , Factores de Transcripción Activadores/genética , Factores de Transcripción Activadores/metabolismo , Animales , Western Blotting , Células COS , Chlorocebus aethiops , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Técnica del Anticuerpo Fluorescente , Humanos , Células Jurkat , Microscopía Confocal , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/análisis , Factores de Transcripción/genética , Activación Transcripcional , Transfección , Factor de Necrosis Tumoral alfa/biosíntesis , Células VeroRESUMEN
African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-kappaB. Thus, transient transfection of the viral IAP increases the activity of an NF-kappaB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-kappaB-dependent gene. NF-kappaB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-kappaB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-kappaB activity seems to be the consequence of higher IkappaB kinase (IKK) basal activity in these cells. The NF-kappaB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2.
Asunto(s)
Virus de la Fiebre Porcina Africana/metabolismo , Regulación de la Expresión Génica , Proteínas de Insectos/metabolismo , FN-kappa B/metabolismo , Virus de la Fiebre Porcina Africana/genética , Animales , Humanos , Proteínas I-kappa B/metabolismo , Proteínas Inhibidoras de la Apoptosis , Proteínas de Insectos/genética , Células Jurkat , Proteínas/metabolismo , Factor 2 Asociado a Receptor de TNF , TransfecciónRESUMEN
Permissive Vero cells develop apoptosis, as characterized by DNA fragmentation, caspases activation, cytosolic release of mitochondrial cytochrome c, and flow cytometric analysis of DNA content, upon infection with African swine fever virus (ASFV). To determine the step in virus replication that triggers apoptosis, we used UV-inactivated virus, inhibitors of protein and nucleic acid synthesis, and lysosomotropic drugs that block virus uncoating. ASFV-induced apoptosis was accompanied by caspase-3 activation, which was detected even in the presence of either cytosine arabinoside or cycloheximide, indicating that viral DNA replication and protein synthesis were not required to activate the apoptotic process. The activation of caspase-3 was released from chloroquine inhibition 2 h after virus absorption, while the infection with UV-inactivated ASFV did not induce the activation of the caspase cascade. We conclude that ASFV induces apoptosis in the infected cell by an intracellular pathway probably triggered during the process of virus uncoating.
Asunto(s)
Virus de la Fiebre Porcina Africana/fisiología , Apoptosis , Replicación Viral/fisiología , Animales , Chlorocebus aethiops , Porcinos , Células Vero , Proteínas Virales/biosíntesisRESUMEN
Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. We have studied the function of p53 in African swine fever virus (ASFV) infection by determining the expression and activity of this transcription factor in infected cells. p53 levels are increased at early times of infection and are maintained throughout the infectious cycle. The protein is transcriptionally active, stabilized by phosphorylation, and localized in the nucleus. p53 induces the expression of p21 and Mdm2. Strikingly, these two proteins are located at the cytoplasmic virus factories. The retention of Mdm2 at the factory may represent a viral mechanism to prevent p53 inactivation by the protein. The expression of apoptotic proteins, such as Bax or active caspase-3, is also increased following ASFV infection, although the increase in caspase-3 does not appear to be, at least exclusively, p53 dependent. Bax probably plays a role in the induction of apoptosis in the infected cells, as suggested by the release of cytochrome c from the mitochondria. The significance of p21 induction and localization is discussed in relation to the shutoff of cellular DNA synthesis that is observed in ASFV-infected cells.
Asunto(s)
Virus de la Fiebre Porcina Africana/patogenicidad , Apoptosis , Regulación de la Expresión Génica , Proteína p53 Supresora de Tumor/metabolismo , Animales , Núcleo Celular/metabolismo , Chlorocebus aethiops , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor/genética , Células Vero/virologíaRESUMEN
Cyclooxygenase-2 is transiently induced upon cell activation or viral infections, resulting in inflammation and modulation of the immune response. Here we report that A238L, an African swine fever virus protein, efficiently inhibits cyclooxygenase-2 gene expression in Jurkat T cells and in virus-infected Vero cells. Transfection of Jurkat cells stably expressing A238L with cyclooxygenase-2 promoter-luciferase constructs containing 5'-terminal deletions or mutations in distal or proximal nuclear factor of activated T cell (NFAT) response elements revealed that these sequences are involved in the inhibition induced by A238L. Overexpression of a constitutively active version of the calcium-dependent phosphatase calcineurin or NFAT reversed the inhibition mediated by A238L on cyclooxygenase-2 promoter activation, whereas overexpression of p65 NFkappaB had no effect. A238L does not modify the nuclear localization of NFAT after phorbol 12-myristate 13-acetate/calcium ionophore stimulation. Moreover, we show that the mechanism by which the viral protein down-regulates cyclooxygenase-2 activity does not involve inhibition of the binding between NFAT and its specific DNA sequences into the cyclooxygenase-2 promoter. Strikingly, A238L dramatically inhibited the transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFAT1. Taken together, these data indicate that A238L down-regulates cyclooxygenase-2 transcription through the NFAT response elements, being NFAT-dependent transactivation implicated in this down-regulation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Proteínas Nucleares/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales/fisiología , Transporte Activo de Núcleo Celular , Animales , Western Blotting , Calcineurina/metabolismo , Calcio/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Eliminación de Gen , Humanos , Ionóforos/farmacología , Células Jurkat , Luciferasas/metabolismo , Proteínas de la Membrana , Microscopía Confocal , Microscopía Fluorescente , Factores de Transcripción NFATC , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Elementos de Respuesta , Acetato de Tetradecanoilforbol/farmacología , Activación Transcripcional , Transfección , Células VeroRESUMEN
The open reading frame EP153R of African swine fever virus (ASFV) encodes a nonessential protein that has been involved in the hemadsorption process induced in virus-infected cells. By the use of a virus deletion mutant lacking the EP153R gene, we have detected, in several virus-sensitive cells, increased levels of caspase-3 and cell death as compared with those obtained after infection with the parental BA71V strain. Both transient and stable expression of the EP153R gene in Vero or COS cells resulted in a partial protection of the transfected lines from the apoptosis induced in response to virus infection or external stimuli. The presence of gene EP153R resulted in a reduction of the transactivating activity of the cellular protein p53 in Vero cell cultures in which apoptosis was induced by virus infection or staurosporine treatment. This is to our knowledge the first description of a viral C-type lectin with anti-apoptotic properties.