RESUMEN
The predicted extracellular domain of the CD2v protein of African swine fever virus (ASFV) shares significant similarity to that of the CD2 protein in T cells but has a unique cytoplasmic domain of unknown function. Here we have shown that CD2v is expressed as a glycoprotein of approximately 105 kDa in ASFV-infected cells. In the absence of an extracellular ligand, the majority of CD2v appears to localize to perinuclear membrane compartments. Furthermore, we have shown using the yeast two-hybrid system and by direct binding studies that the cytoplasmic tail of CD2v binds to the cytoplasmic adaptor protein SH3P7 (mAbp1, HIP55), which has been reported to be involved in diverse cellular functions such as vesicle transport and signal transduction. A cDNA clone encoding a variant form of SH3P7 could also be identified and was found to be expressed in a wide range of porcine tissues. Deletion mutagenesis identified proline-rich repeats of sequence PPPKPC in the ASFV CD2v protein to be necessary and sufficient for binding to the SH3 domain of SH3P7. In ASFV-infected cells, CD2v and SH3P7 co-localized in areas surrounding the perinuclear virus factories. These areas also stained with an antibody that recognizes a Golgi network protein, indicating that they contained membranes derived from the Golgi network. Our data provide a first molecular basis for the understanding of the immunomodulatory functions of CD2v in ASFV-infected animals.
Asunto(s)
Virus de la Fiebre Porcina Africana/fisiología , Antígenos CD2/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Virales/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD2/química , Antígenos CD2/genética , Chlorocebus aethiops , Citoplasma/metabolismo , Eliminación de Gen , Glicosilación , Datos de Secuencia Molecular , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Células Vero , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-kappaB-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Chlorocebus aethiops , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Fragmentos de Péptidos/metabolismo , Porcinos , Células Vero , Proteínas Virales/metabolismoRESUMEN
The NH(2)-terminal end of a protein, named SMCp, which contains an ARID (A/T rich interaction domain) DNA binding domain and is similar to the mammalian SMCY/SMCX proteins and retinoblastoma binding protein 2, was shown to bind the African swine fever virus encoded ubiquitin conjugating enzyme (UBCv1) using the yeast two hybrid system and in in vitro binding assays. Antisera raised against the SMCp protein were used to show that the protein is present in the cell nucleus. Immunofluorescence showed that although UBCv1 is present in the nucleus in most cells, in some cells it is in the cytoplasm, suggesting that it shuttles between the nucleus and cytoplasm. The interaction and co-localisation of UBCv1 with SMCp suggest that SMCp may be a substrate in vivo for the enzyme.
Asunto(s)
Virus de la Fiebre Porcina Africana/enzimología , Ligasas/metabolismo , Proteínas/metabolismo , Enzimas Ubiquitina-Conjugadoras , Proteínas Virales , Virus de la Fiebre Porcina Africana/metabolismo , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Núcleo Celular/virología , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Demetilasas , N-Metiltransferasa de Histona-Lisina , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes , Unión Proteica , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
PCR analysis of the genomes of 18 different African swine fever virus (ASFV) isolates showed that the I14L open reading frame (ORF) was present as either a long form or short form in all of the isolates. Sequencing of the ORF from eight isolates confirmed that both forms of the ORF were well conserved. Antisera raised against the I14L protein identified the long form of the protein as a 21 kDa protein expressed late during ASFV infection. Immunofluorescent analysis of transiently expressed haemagglutinin-tagged forms of the I14L protein showed that the long form of the protein localized predominantly to the nucleus and within the nucleoli. In contrast, although the short form of the protein was also present predominantly in the nucleus, it did not localize to the nucleoli. Deletion of the N-terminal 14 amino acids from the long form of the I14L protein, which includes a high proportion of basic Arg/Lys residues, abolished the specific nucleolar localization of the protein, although the protein was still present in the nucleus. Addition of this 14 amino acid sequence to beta-galactosidase or replacement of the N-terminal 14 amino acids of the I14L short form with those from the long form directed both of these modified proteins to the nucleolus. This indicates that this 14 amino acid sequence contains all the signals required for nucleolar localization.
Asunto(s)
Virus de la Fiebre Porcina Africana/metabolismo , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/crecimiento & desarrollo , Virus de la Fiebre Porcina Africana/patogenicidad , Secuencia de Aminoácidos , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Macrófagos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , Homología de Secuencia de Aminoácido , Simplexvirus/química , Simplexvirus/genética , Simplexvirus/patogenicidad , Porcinos , Factores de Tiempo , Transfección , Proteínas Virales/química , Virulencia/genéticaRESUMEN
The transcription factor NFAT (nuclear factor of activated T cells) controls the expression of many immunomodulatory proteins. African swine fever virus inhibits proinflammatory cytokine expression in infected macrophages, and a viral protein A238L was found to display the activity of the immunosuppressive drug cyclosporin A by inhibiting NFAT-regulated gene transcription in vivo. This it does by binding the catalytic subunit of calcineurin and inhibiting calcineurin phosphatase activity.