Poxviral B1 Kinase Overcomes Barrier to Autointegration Factor, a Host Defense against Virus Replication

Barrier to autointegration factor (BAF) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mito-sis. Herein, we demonstrate a cytoplasmic role for BAF in host defense during poxviral infections. Vaccinia is the prototypic poxvirus, a family of DNA viruses that replicate ex-clusively in the cytoplasm of infected cells. Mutations in the vaccinia B1 kinase (B1) com-promise viral DNA replication, but the mechanism by which B1 achieves this has remained elusive. We now show that BAF acts as a potent inhibitor of poxvirus replication unless its DNA-binding activity is blocked by B1-mediated phosphorylation. These data position BAF as the effector of an innate immune response that prevents replication of exogenous viral DNA in the cytoplasm. To enable the virus to evade this defense, the poxviral B1 has evolved to usurp a signaling pathway employed by the host cell

The Vaccinia-related Kinases Phosphorylate the N\u27 Terminus of BAF, Regulating Its Interaction with DNA and Its Retention in the Nucleus

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N\u27 terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain–containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture

The Vaccinia-related Kinases Phosphorylate the N\u27 Terminus of BAF, Regulating Its Interaction with DNA and Its Retention in the Nucleus

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N\u27 terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain–containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture

Functional characterization of the vaccinia virus I5 protein

The I5L gene is one of ~90 genes that are conserved throughout the chordopoxvirus family, and hence are presumed to play vital roles in the poxvirus life cycle. Previous work had indicated that the VP13 protein, a component of the virion membrane, was encoded by the I5L gene, but no additional studies had been reported. Using a recombinant virus that encodes an I5 protein fused to a V5 epitope tag at the endogenous locus (vI5V5), we show here that the I5 protein is expressed as a post-replicative gene and that the ~9 kDa protein does not appear to be phosphorylated in vivo. I5 does not appear to traffic to any cellular organelle, but ultrastructural and biochemical analyses indicate that I5 is associated with the membranous components of assembling and mature virions. Intact virions can be labeled with anti-V5 antibody as assessed by immunoelectron microscopy, indicating that the C' terminus of the protein is exposed on the virion surface. Using a recombinant virus which encodes only a TET-regulated copy of the I5V5 gene (vΔindI5V5), or one in which the I5 locus has been deleted (vΔI5), we also show that I5 is dispensable for replication in tissue culture. Neither plaque size nor the viral yield produced in BSC40 cells or primary human fibroblasts are affected by the absence of I5 expression

Identification and characterization of the orf virus type I topoisomerase

AbstractVaccinia virus (VV) and Shope fibroma virus (SFV), representatives of the orthopox and leporipox genera, respectively,encode type I DNA topoisomerases. Here we report that the 957-nt F4R open reading frame of orf virus (OV), a representative of the parapox genus, is predicted to encode a 318-aa protein with extensive homology to these enzymes. The deduced amino acid sequence of F4R has 54.7 and 50.6% identity with the VV and SFV enzymes, respectively. One hundred forty amino acids are predicted to be conserved in all three proteins. The F4R protein was expressed in Escherichia coli under the control of an inducible T7 promoter, partially purified, and shown to be a bona fide type I topoisomerase. Like the VV enzyme, the OV enzyme relaxed negatively supercoiled DNA in the absence of divalent cations or ATP and formed a transient covalent intermediate with cleaved DNA that could be visualized by SDS-PAGE. Both the noncovalent and covalent protein/DNA complexes could be detected in an electrophoretic mobility shift assay. The initial PCR used to prepare expression constructs yielded a mutant allele of the OV topoisomerase with a G-A transition at nt 677 that was predicted to replace a highly conserved Tyr residue with a Cys. This allele directed the expression of an enzyme which retained noncovalent DNA binding activity but was severely impaired in DNA cleavage and relaxation. Incubation of pUC19 DNA with the wild-type OV or VV enzyme yielded an indistinguishable set of DNA cleavage fragments, although the relative abundance of the fragments differed for the two enzymes. Using a duplex oligonucleotide substrate containing the consensus site for the VV enzyme, we demonstrated that the OV enzyme also cleaved efficiently immediately downstream of the sequence CCCTT↓

Poxvirus Bioinformatics Resource Center: a comprehensive Poxviridae informational and analytical resource

The Poxvirus Bioinformatics Resource Center (PBRC) has been established to provide informational and analytical resources to the scientific community to aid research directed at providing a better understanding of the Poxviridae family of viruses. The PBRC was specifically established as the result of the concern that variola virus, the causative agent of smallpox, as well as related viruses, might be utilized as biological weapons. In addition, the PBRC supports research on poxviruses that might be considered new and emerging infectious agents such as monkeypox virus. The PBRC consists of a relational database and web application that supports the data storage, annotation, analysis and information exchange goals of the project. The current release consists of over 35 complete genomic sequences of various genera, species and strains of viruses from the Poxviridae family. Sequence and annotation information for these viruses has been obtained from sequences publicly available from GenBank as well as sequences not yet deposited in GenBank that have been obtained from ongoing sequencing projects. In addition to sequence data, the PBRC provides comprehensive annotation and curation of virus genes; analytical tools to aid in the understanding of the available sequence data, including tools for the comparative analysis of different virus isolates; and visualization tools to help better display the results of various analyses. The PBRC represents the initial development of what will become a more comprehensive Viral Bioinformatics Resource Center for Biodefense that will be one of the National Institute of Allergy and Infectious Diseases' ‘Bioinformatics Resource Centers for Biodefense and Emerging or Re-Emerging Infectious Diseases’. The PBRC website is available at http://www.poxvirus.org

Hasta transformarlo todo Ciberactivismo en Instagram Estudio de casos

Trabajo Final para optar al grado académico de Licenciatura en Comunicación Social, Universidad Nacional de CórdobaEn el presente Trabajo Final de Grado, abordamos el tema de los ciberactivismos en la red social Instagram. Profundizamos en las configuraciones discursivas que expresan estas nuevas formas de activismo digital, en tanto que expresión política contemporánea. En la actualidad, las redes sociales resultan un espacio de expansión, crecimiento y circulación de nuevas prácticas comunicativas y discursivas de carácter político y este trabajo indaga en torno al despliegue del ciberactivismo en esta plataforma. Damos cuenta de ciertas particularidades que adquieren perfiles de instagramers que devienen sujetos políticos referentes de causas y movimientos emergentes. El trabajo recibe los aportes del pensamiento teórico de los Estudios Mediales y de las discusiones en torno a la tensión que surge en el entramado de ciberculturas, nuevas subjetividades y activismo político en época de redes sociales. Metodológicamente trabajamos a partir de una exploración y reconocimiento del campo y luego desde la perspectiva sociosemiótica, ya que mediante el análisis discursivo se indaga en torno a posibilidades enunciativas, consolidación de destinatarios y entidades, siendo los componentes de la hegemonía, una entrada central y operativa para dicho análisis. Las materias significantes abordadas pertenecen a las cuentas de @brenda.mato, @nutriloca y @sol_despeinada y refieren a causas tales como: body positive, veganismo-antiespecismo y feminismo. Dada la novedad del fenómeno analizado y los escasos antecedentes metodológicos de análisis discursivo en la red social Instagram, este abordaje resulta un desafío importante que permite la exploración de este espacio y momento del discurso social contemporáneo.Fil: Gómez, María Luz. Universidad Nacional de Córdoba. Facultad de Ciencias de la Comunicación; Argentina.Fil: Simioni, Lucía. Universidad Nacional de Córdoba. Facultad de Ciencias de la Comunicación; Argentina.Fil: Traktman, Noelia Andrea. Universidad Nacional de Córdoba. Facultad de Ciencias de la Comunicación; Argentina

Distribution of sources and transfer of tools made of quartz from the Sierras of Córdoba, Argentina

En este trabajo se presentan los resultados del análisis de fuentes de cuarzo, del material recuperado en tres canteras taller, denominadas Arroyo La Mina 1 (CALM1), Arroyo El Vigilante 1 (CAEV1) y Arroyo El Tabaquillo 1 (CAET1) ubicadas en las sierras de Córdoba y de una colección de instrumentos procedente de cuatro sitios de la microrregión del sur de Punilla. La metodología consistió, por un lado, en prospecciones, relevamientos y recolecciones superficiales en los sitios cantera taller; y, por el otro lado, en el análisis lítico a partir de aproximaciones tecno- morfológicas. El estudio de las fuentes de cuarzo demuestra la importancia que ha tenido este recurso en la confección de instrumentos y bifaces en el pasado. Esta materia prima, que incluye la variedad hialina o cristal de roca, alcanza una distribución hacia las zonas de fondo de valle, manifestada por la presencia de puntas de proyectil características del Holoceno tardío final.This paper presents the results of the analysis carried out on quartz sources of the material recovered in three workshop qu arries: Arroyo La Mina 1 (CALM1), Arroyo El Vigilante 1 (CAEV1) and Arroyo El Tabaquillo 1 (CAET1) located in Córdoba ranges, and of a collection of tools from four sites of the micro region of southern Punilla. The methodology consisted in, on the one hand, field surveys and surface collections in quarry-workshop areas and on the other hand, in lithic analysis from techno-morphological approaches. The study of quartz sources reveals the importance that this resource has had in the manufacture of tools and bifaces in the past. This raw material, that includes the hyaline variety or rock crystal, reaches a distribution towards the valley floor areas displayed by the presence of projectile points typical of the final late Holocene.Sociedad Argentina de Antropologí

Classification of groups and petrographic analysis: towards a characterization of the pottery assemblies of the Copacabana River Basin (Ischilín, Córdoba, Argentina)

En este artículo se exponen los resultados obtenidos a partir del análisis macro y microscópico sobre los conjuntos cerámicos de cuatro sitios de la cuenca del río Copacabana (Ischilín, Córdoba, Argentina). El objetivo es indagar sobre las distintas formas de manufactura de la alfarería. Se elaboró una tipología cerámica y, seguidamente, se testeó la variabilidad de los grupos identificados, mediante la implementación de técnicas petrográficas. Basándose en estos primeros análisis, se identificaron cuatro grupos petrográficos, que indican una diferencia en la incorporación del antiplástico en las pastas cerámicas y, por lo tanto, se infieren distintas modalidades de hacer.In this article we present the results obtained from the macro and microscopic analysis of the pottery assemblies of four sites located in the Copacabana river basin (Ischilín, Córdoba, Argentina). The purpose of this study is to investigate the different ways of manufacturing ceramics. Firstly, a typology was elaborated, and then the variability of the identified groups was tested trough the implementation of petrographic techniques. Based on these first analyzes, four petrographic groups were identified which indicate a difference in the addition of antiplastic in ceramic pastes and, therefore, different ways of making pottery are inferred.Fil: Traktman, Macarena Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Antropología de Córdoba. Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades. Instituto de Antropología de Córdoba; ArgentinaFil: Sario, Gisela Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Antropología de Córdoba. Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades. Instituto de Antropología de Córdoba; ArgentinaFil: Salvatore, Marcos Aníbal. Comisión Nacional de Energía Atómica; ArgentinaFil: Anzil, Patricia Andrea. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Lipid Membranes in Poxvirus Replication

Poxviruses replicate in the cytoplasm, where they acquire multiple lipoprotein membranes. Although a proposal that the initial membrane arises de novo has not been substantiated, there is no accepted explanation for its formation from cellular membranes. A subsequent membrane-wrapping step involving modified trans-Golgi or endosomal cisternae results in a particle with three membranes. These wrapped virions traverse the cytoplasm on microtubules; the outermost membrane is lost during exocytosis, the middle one is lost just prior to cell entry, and the remaining membrane fuses with the cell to allow the virus core to enter the cytoplasm and initiate a new infection