Cubeta para la incubación y lavado de anticuerpos

Cubeta para la incubación y lavado de anticuerpos. La cubeta comprende un cuerpo (1) lavable, inerte y no poroso, que está dotado de unos pocillos (2) ubicados en su interior, cuyo número y tamaño puede variar en función del requerimiento experimental. En dichos pocillos (2) se introducen preferiblemente fragmentos o tiras (3) de nitrocelulosa, que contienen proteínas que han sido previamente transferidas desde un gel de poliacrilamida utilizando un campo eléctrico. Esta cubeta tiene como principal aplicación la técnica Western Blot, que permite la detección y localización de cada una de las proteínas mediante la incubación de estas tiras (3) con anticuerpos específicos frente a las proteínas contenidas en ellas.Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Cubeta para la incubación y lavado de anticuerpos

Cubeta para la incubación y lavado de anticuerpos. La cubeta comprende un cuerpo (1) lavable, inerte y no poroso, que está dotado de unos pocillos (2) ubicados en su interior, cuyo número y tamaño puede variar en función del requerimiento experimental. En dichos pocillos (2) se introducen preferiblemente fragmentos o tiras (3) de nitrocelulosa, que contienen proteínas que han sido previamente transferidas desde un gel de poliacrilamida utilizando un campo eléctrico. Esta cubeta tiene como principal aplicación la técnica Western Blot, que permite la detección y localización de cada una de las proteínas mediante la incubación de estas tiras (3) con anticuerpos específicos frente a las proteínas contenidas en ellas.Peer reviewedConsejo Superior de Investigaciones Científicas (España)B1 Patente sin examen previ

CD2v interacts with Adaptor Protein AP-1 during African swine fever infection

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.This work was supported by Ministerio de Ciencia e Innovación of Spain, BFU2010-17794 (YR); European Community’s Seventh Framework Programme, KBBE.2012.1.3-02-ASFORCE (YR). Ricardo Madrid was funded by an Amarauto research program and by FIS-641 PS09/01386.Peer Reviewe

Cubeta para la incubación y lavado de anticuerpos

[EN] The vessel comprises a non-porous, inert, washable body (1) provided with wells (2) located within, the number and size of which wells may vary as a function of experimental requirements. Said wells (2) preferably receive fragments or strips (3) of nitrocellulose that contain proteins previously transferred from a polyacrylamide gel using an electric field. The main use of this vessel is in Western blotting, which allows the detection and location of each protein by means of the incubation of said strips (3) with antibodies specific for the proteins contained therein.[ES] La cubeta comprende un cuerpo (1) lavable, inerte y no poroso, que está dotado de unos pocillos (2) ubicados en su interior, cuyo número y tamaño puede variar en función del requerimiento experimental. En dichos pocillos (2) se introducen preferiblemente fragmentos o tiras (3) de nitrocelulosa, que contienen proteínas que han sido previamente transferidas desde un gel de poliacrilamida utilizando un campo eléctrico. Esta cubeta tiene como principal aplicación la técnica Western Blot, que permite la detección y localización de cada una de las proteínas mediante la incubación de estas tiras (3) con anticuerpos específicos frente a las proteínas contenidas en ellas.Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

The African swine fever virus virion membrane protein pE248R is required for virus infectivity and an early postentry event

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.This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (BFU2007-61647) and the Wellcome Trust (075813/C/04/Z) and by an institutional grant from Fundación Ramón Areces. M. Redrejo-Rodríguez was a Fellow of the Ministerio de Ciencia e Innovación

Regulation of Host Translational Machinery by African Swine Fever Virus

African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host’s defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2a, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV protein production in infected cells.This work was supported by Grants from Laboratorios Esteve, Ministerio de Educacio´n y Ciencia BFU2007-63110 and BFU2006-02182, and by an Institutional grant from the Fundacio´n Ramo´n Areces. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

African swine fever virus controls the host transcription and cellular machinery of protein synthesis

Throughout a viral infection, the infected cell reprograms the gene expression pattern in order to establish a satisfactory antiviral response. African swine fever virus (ASFV), like other complex DNA viruses, sets up a number of strategies to evade the host's defense systems, such as apoptosis, inflammation and immune responses. The capability of the virus to persist in its natural hosts and in domestic pigs, which recover from infection with less virulent isolates, suggests that the virus displays effective mechanisms to escape host defense systems. ASFV has been described to regulate the activation of several transcription factors, thus regulating the activation of specific target genes during ASFV infection.Whereas some reports have concerned about anti-apoptotic ASFV genes and the molecular mechanisms by which ASFV interferes with inducible gene transcription and immune evasion, less is yet known regarding how ASFV regulates the translational machinery in infected cells, although a recent report has shown a mechanism for favored expression of viral genes based on compartmentalization of viral mRNA and ribosomes with cellular translation factors within the virus factory.The viral mechanisms involved both in the regulation of host genes transcription and in the control of cellular protein synthesis are summarized in this review. © 2012 Elsevier B.V.Spanish Ministerio de Ciencia e Innovación (BFU2007-63110/BFU2010-17794); European Community’s Seventh Framework Programme (FP7/2007–2013); KBBE-211691-ASFRISK; Fundación Ramón Areces.Peer Reviewe

African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.This work was supported by grants from the Comunidad Autónoma de Madrid (08.8/0005/1997), the Ministerio de Educacio´n y Cultura (Programa Sectorial de Promocio´n General del Conocimiento and Programa Nacional de Investigación y Desarrollo Agrario; PB96-0902- CO2-01 and AGF98-1352-CE), and the European Commission (FAIR5-PL97-3441) and by an institutional grant from the Fundación Ramón Areces. B. Cubelos was a fellow of the Comunidad Autónoma de Madrid

The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway

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 NFκB 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.This work was supported in part by grants from Ministerio de Ciencia y Tecnología (Grants BMC2000-1485 and AGL2002-10220-E), the European Commission (QLRT-2000-02216), and by an institutional grant from the Fundación Ramón Areces

Antiviral activity of lauryl gallate against animal viruses

Suplemmentary material is avalaible at the end of the paperThe effect of the anti-tumoral drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (Herpes simplex and Vaccinia) and RNA (Influenza, Porcine transmissible gastroenteritis and Sindbis) viruses, involved in animal and human diseases, is analyzed. Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1-10 μM), reducing the titres from 3 to more than 5 log. units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption, completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5 to 8 hpi. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug, but, however, the early viral protein synthesis and the virus-mediated increasing of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells, and, furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. The overall results likely indicate that a cellular factor/function might be the target of the antiviral action of alkyl gallatesThis work was supported by grants from Ministerio de Educación y Ciencia (Nº BFU2004-00298/BMC), Laboratorios del Dr. Esteve, and by an institutional grant from the Fundación Ramón Areces. C.Hurtado was a fellow from Fundación Ramón Areces. A.G. Granja was funded by Centro de Investigación en Sanidad Animal (CISA).Peer reviewe