Biomedical subjects

E Viñuela

Publications and source records attributed to E Viñuela.

At least 19 recordsLinked to original sources

PubMed2004

Influence of azadirachtin, a botanical insecticide, on Chrysoperla carnea (Stephens) reproduction: toxicity and ultrastructural approach.

A commercial formulation of azadirachtin (Align) was tested on Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) adults to determine its effects on reproduction. Three methods of exposure were carried out in the laboratory (residual, direct contact and ingestion) with concentrations close to the maximum field recommended concentration in Spain (48 mg AI/liter). Align proved to be harmless to newly emerged adults irrespective of the mode of exposure. Nevertheless, fecundity was reduced in a reversible way in females that have ingested azadiracthin after the onset of oviposition. Fertility was not different from that of controls in all treatments. Additional tests showed that males were not involved in the reduction of oviposition. Electron microscopy studies pointed out that Align interfered with vitellogenin synthesis and/or its uptake by developing oocytes; thus, growing follicles in treated females were significantly smaller that those of the controls. The role of azadirachtin as a compound that may influence several hormonal and protein titers involved in reproduction is also discussed.

Animals↗

PubMed2003

Topical toxicity of two acetonic fractions of Trichilia havanensis Jacq. and four insecticides to larvae and adults of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae).

The toxicity of botanical origin compounds such as two acetonic fractions of the seed kernels of the Meliacea Trichilia havanensis Jacq with insecticide properties (azadirone (F12) and the mixture F18 [1,7+3,7-di-O-acethylhavanensin (4:1)], three insecticides commercially available (imidacloprid, natural pyrethrins+PBO, triflumuron) and phloxine B, were tested in the laboratory. Topical bioassays using third instar and newly emerged adults of the lacewing Chrysoperla carnea (Stephens) at the maximum field recommended rate in Spain for commercials and at 1,000 ppm of active ingredient for T. havanensis acetone fractions and phloxine-B, were carried out. Imidacloprid and triflumuron were very toxic to third instar larvae inhibiting adult emergence, being the rest of insecticides harmless. Fecundity and fertility were not affected by the non-toxic compounds. Concerning adults, only imidacloprid and natural pyrethrins killed them significantly 24 hours after treatment. Phloxine B, triflumuron and T. havanensis fractions were harmless and did not cause any effect on fecundity and fertility with the exception of triflumuron, which reduced considerably the egg hatch. It can be concluded that T. havanensis acetonic fractions and phloxine B were non-toxic to larvae and adults of C. carnea when treated topically, whereas triflumuron, natural pyrethrins and imidacloprid affected one or more of the evaluated parameters under our conditions.

Acetone↗

PubMed2001

African swine fever virus structural protein pE120R is essential for virus transport from assembly sites to plasma membrane but not for infectivity.

This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lac repressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.

African Swine Fever Virus↗

PubMed2000

African swine fever virus EP153R open reading frame encodes a glycoprotein involved in the hemadsorption of infected cells.

The open reading frame EP153R, located within the EcoRI E' fragment of the African swine fever (ASF) virus genome, is predicted to encode a membrane protein of 153 amino acids that presents significant homology to the N-terminal region of several CD44 molecules. EP153R contains multiple putative sites for N-glycosylation, phosphorylation, and myristoylation, a central transmembrane region, a C-type animal lectin-like domain, and a cell attachment sequence. Transcription of EP153R takes place at both early and late times during the virus infection. The disruption of the gene, achieved by insertion of the marker gene LacZ within EP153R, did not change either the in vitro virus growth rate or the virus-sensitive/resistant condition of up to 17 established cell lines, but abrogated the hemadsorption phenomenon induced in ASF virus-infected cells. As the sequence and expression of the ASF virus protein pEP402R, a CD2 homolog responsible for the adhesion of erythrocytes to susceptible cells, was unaffected in cultures infected with the EP153R deletion mutant, we conclude that the gene EP153R is needed to induce and/or maintain the interaction between the viral CD2 homolog and its corresponding cell receptor.

African Swine Fever Virus↗

PubMed2000

Characterization of the african swine fever virus protein p49: a new late structural polypeptide.

The open reading frame B438L, located within the EcoRI B fragment of the African swine fever virus genome, is predicted to encode a protein of 438 amino acids with a molecular mass of 49.3 kDa. It presents a cell attachment RGD (Arg-Gly-Asp) motif but no other significant similarity to protein sequences in databases. Northern blot and primer extension analysis showed that B438L is transcribed only at late times during virus infection. The B438L gene product has been expressed in Escherichia coli, purified and used as an antigen for antibody production. The rabbit antiserum specific for pB438L recognized a protein of about 49 kDa in virus-infected cell extracts. This protein was synthesized late in infection by all the virus strains tested, was located in cytoplasmic virus factories and appeared as a structural component of purified virus particles.

African Swine Fever Virus↗

PubMed2000

Structure of African swine fever virus late promoters: requirement of a TATA sequence at the initiation region.

A number of mutations, including deletions, linker scan substitutions, and point mutations, were performed in the promoter of the late African swine fever virus (ASFV) gene coding for the capsid protein p72. The consequences of the mutations in terms of promoter activity were analyzed by luciferase assays using plasmids transfected into infected cells. The results showed that the promoter function is contained between nucleotides -36 and +5 relative to the transcription initiation site. Moreover, two major essential regions for promoter activity, centered at positions -13 and +3, were located along the 41-bp sequence, the latter mapping in the transcription start site. Sequence alignment with other ASFV late promoters showed homology in the region of transcriptional initiation, where the presence of the sequence TATA was observed in most of the promoters. Substitution of these four residues in three other late viral promoters strongly reduced their respective activities. These results show that cis-acting control elements of ASFV p72 gene transcription are restricted to a short sequence of about 40 bp and suggest that transcription of late genes is initiated around a TATA sequence that would function as an initiator element.

African Swine Fever Virus↗

PubMed1999

The African swine fever virus prenyltransferase is an integral membrane trans-geranylgeranyl-diphosphate synthase.

In a previous study, it was shown that the protein encoded by the gene B318L of African swine fever virus (ASFV) is a trans-prenyltransferase that catalyzes in vitro the condensation of farnesyl diphosphate and isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and longer chain prenyl diphosphates (Alejo, A., Yáñez, R. J., Rodríguez, J. M., Viñuela, E., and Salas, M. L. (1997) J. Biol. Chem. 272, 9417-9423). To investigate the in vivo function of the viral enzyme, we have determined, in this work, its subcellular localization and activity in cell extracts. Two systems were used in these studies: cells infected with ASFV and cells infected with a recombinant pseudo-Sindbis virus carrying the complete B318L gene. In this latter system, the trans-prenyltransferase was found to colocalize with the endoplasmic reticulum marker protein-disulfide isomerase, whereas in cells infected with ASFV, the viral enzyme was present in cytoplasmic viral assembly sites, associated with precursor viral membranes derived from the endoplasmic reticulum. In addition, after subcellular fractionation, the viral enzyme partitioned into the membrane fraction. Extraction of membrane proteins with alkaline carbonate and Triton X-114 indicated that the ASFV enzyme behaved as an integral membrane protein. The membrane enzyme synthesized predominantly all-trans-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate. These results indicate that the viral B318L protein is a trans-geranylgeranyl-diphosphate synthase, being the only enzyme of this type that is known to have a membrane localization.

African Swine Fever Virus↗

PubMed1999

Replication of African swine fever virus DNA in infected cells.

We have examined the ultrastructural localization of African swine fever virus DNA in thin-sections of infected cells by in situ hybridization and autoradiography. Virus-specific DNA sequences were found in the nucleus of infected Vero cells at early times in the synthesis of the viral DNA, forming dense foci localized in proximity to the nuclear membrane. At later times, the viral DNA was found exclusively in the cytoplasm. Electron microscopic autoradiography of African swine fever virus-infected macrophages showed that the nucleus is also a site of viral DNA replication at early times. These results provide further evidence of the existence of nuclear and cytoplasmic stages in the synthesis of African swine fever virus DNA. On the other hand, alkaline sucrose sedimentation analysis of the replicative intermediates synthesized in the nucleus and cytoplasm of infected macrophages showed that small DNA fragments ( approximately 6-12S) were synthesized in the nucleus at an early time, whereas at later times, larger fragments of approximately 37-49S were labeled in the cytoplasm. Pulse-chase experiments demonstrated that these fragments are precursors of the mature cross-linked viral DNA. The formation of dimeric concatemers, which are predominantly head-to-head linked, was observed by pulsed-field electrophoresis and restriction enzyme analysis at intermediate and late times in the replication of African swine fever virus DNA. Our findings suggest that the replication of African swine fever virus DNA proceeds by a de novo start mechanism with the synthesis of small DNA fragments, which are then converted into larger size molecules. Ligation or further elongation of these molecules would originate a two-unit concatemer with dimeric ends that could be resolved to generate the genomic DNA by site-specific nicking, rearrangement, and ligation as has been proposed in the de novo start model of Baroudy et al. (B. M. Baroudy, S. Venkatesam, and B. Moss, 1982, Cold Spring Harbor Symp. Quant. Biol. 47, 723-729) for the replication of vaccinia virus DNA.

African Swine Fever Virus↗

PubMed1999

Virus-specific cell receptors are necessary, but not sufficient, to confer cell susceptibility to African swine fever virus.

The entry of African swine fever (ASF) virus into Vero cells and swine macrophages is mediated by saturable binding sites located in the plasma membrane, which have been related, as in other virus-cell systems, to the sensitivity of the cell to the virus. In order to define this correlation, we have analyzed up to 16 cell lines derived from different species for their sensitivity to virus infection, to determine the step in the virus infective cycle that was blocked in each resistant cell, the presence of saturable cell receptors and the percentage of bound and internalized virus in these cells. Specific ASF virus receptors were found in different quantities in many sensitive and resistant cell lines. The most restricted cells showed a reduced efficiency of virus binding and virus internalization, as well as a lower amount of cell receptors for the virus attachment protein p12. Other resistant cells were restricted only after early virus translation or virus DNA replication, proving that the presence of virus-specific receptors may be necessary, but not sufficient, to guarantee the cell permissiveness to the virus, and that the ASF virus infection can be arrested at different steps on the infective cycle.

African Swine Fever Virus↗

PubMed1999

African swine fever virus dUTPase is a highly specific enzyme required for efficient replication in swine macrophages.

The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg(2+), the preferred cation, and has an apparent K(m) for dUTP of 1 microM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vDeltaE165R) has been obtained. Growth kinetics showed that vDeltaE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.

African Swine Fever↗

PubMed1998

Inhibition of nuclear factor kappaB activation by a virus-encoded IkappaB-like protein.

Certain viruses have evolved mechanisms to counteract innate immunity, a host response in which nuclear factor kappaB (NF-kappaB) transcription factors play a central role. African swine fever virus encodes a protein of 28.2 kDa containing ankyrin repeats similar to those of cellular IkappaB proteins, which are inhibitors of NF-kappaB. Transfection of the African swine fever virus IkappaB gene inhibited tumor necrosis factor- or phorbol ester-induced activation of kappaB- but not AP-1-driven reporter genes. Moreover, African swine fever virus IkappaB co-immunoprecipitated with p65 NF-kappaB, and the purified recombinant protein prevented the binding of p65-p50 NF-kappaB proteins to their target sequences in the DNA. NF-kappaB activation induced by tumor necrosis factor, as detected by mobility shift assays or by transfection of kappaB-driven reporter genes, is impaired in African swine fever virus-infected cells. These results indicate that the African swine fever virus IkappaB gene homologue interferes with NF-kappaB activation, likely representing a new mechanism to evade the immune response during viral infection.

African Swine Fever Virus↗

PubMed1998

Vaccinia virus-induced apoptosis in immature B lymphocytes: role of cellular Bcl-2.

Apoptosis is a form of physiological cell death which can be initiated in response to various stimuli including virus infections. We show that vaccinia virus (VV) infection induces apoptosis in an immature B lymphocyte line, WEHI-231. In these cells, several VV-specific proteins were synthesized during the infection, but neither virus production nor viral DNA synthesis were detected. The intracellular levels of the proto-oncogene Bcl-2, which effectively protects cells from programmed cell death, were found to be down-regulated by the VV infection, suggesting that this down-regulation might be involved in the viral induction of apoptosis in WEHI-231 cells. Stable transfectants overexpressing human Bcl-2 were shown to be resistant to the apoptosis produced by the infection, a finding consistent with the proposed role for the down-regulation of endogenous Bcl-2 in VV-induced apoptotic death.

Animals↗

PubMed1998

African swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum.

During the cytoplasmic maturation of African swine fever virus (ASFV) within the viral factories, the DNA-containing core becomes wrapped by two shells, an inner lipid envelope and an outer icosahedral capsid. We have previously shown that the inner envelope is derived from precursor membrane-like structures on which the capsid layer is progressively assembled. In the present work, we analyzed the origin of these viral membranes and the mechanism of envelopment of ASFV. Electron microscopy studies on permeabilized infected cells revealed the presence of two tightly apposed membranes within the precursor membranous structures as well as polyhedral assembling particles. Both membranes could be detached after digestion of intracellular virions with proteinase K. Importantly, membrane loop structures were observed at the ends of open intermediates, which suggests that the inner envelope is derived from a membrane cisterna. Ultraestructural and immunocytochemical analyses showed a close association and even direct continuities between the endoplasmic reticulum (ER) and assembling virus particles at the bordering areas of the viral factories. Such interactions become evident with an ASFV recombinant that inducibly expresses the major capsid protein p72. In the absence of the inducer, viral morphogenesis was arrested at a stage at which partially and fully collapsed ER cisternae enwrapped the core material. Together, these results indicate that ASFV, like the poxviruses, becomes engulfed by a two-membraned collapsed cisterna derived from the ER.

African Swine Fever Virus↗

PubMed1998

Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72.

A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.

African Swine Fever Virus↗

PubMed1998

Migration of mitochondria to viral assembly sites in African swine fever virus-infected cells.

An examination by electron microscopy of the viral assembly sites in Vero cells infected with African swine fever virus showed the presence of large clusters of mitochondria located in their proximity. These clusters surround viral factories that contain assembling particles but not factories where only precursor membranes are seen. Immunofluorescence microscopy revealed that these accumulations of mitochondria are originated by a massive migration of the organelle to the virus assembly sites. Virus infection also promoted the induction of the mitochondrial stress-responsive proteins p74 and cpn 60 together with a dramatic shift in the ultrastructural morphology of the mitochondria toward that characteristic of actively respiring organelles. The clustering of mitochondria around the viral factory was blocked in the presence of the microtubule-disassembling drug nocodazole, indicating that these filaments are implicated in the transport of the mitochondria to the virus assembly sites. The results presented are consistent with a role for the mitochondria in supplying the energy that the virus morphogenetic processes may require and make of the African swine fever virus-infected cell a paradigm to investigate the mechanisms involved in the sorting of mitochondria within the cell.

African Swine Fever Virus↗

PubMed1997

Characterization of an African swine fever virus 20-kDa DNA polymerase involved in DNA repair.

African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3'-5'-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase beta (pol beta), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol beta-type DNA polymerase core, constituted by only the "palm" and "thumb" subdomains. It is worth noting that such an "unfingered" DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.

African Swine Fever Virus↗

PubMed1997

African swine fever virus trans-prenyltransferase.

The present study describes the characterization of an African swine fever virus gene homologous to prenyltransferases. The gene, designated B318L, is located within the EcoRI B fragment in the central region of the virus genome, and encodes a polypeptide with a predicted molecular weight of 35,904. The protein is characterized by the presence of a putative hydrophobic transmembrane domain at the amino end. The gene is expressed at the late stage of virus infection, and transcription is initiated at positions -118, -119, -120, and -122 relative to the first nucleotide of the translation start codon. Protein B318L presents a colinear arrangement of the four highly conserved regions and the two aspartate-rich motifs characteristic of geranylgeranyl diphosphate synthases, farnesyl diphosphate synthases, and other prenyltransferases. Throughout these regions, the percentages of identity between protein B318L and various prenyltransferases range from 28.6 to 48.7%. The gene was cloned in vector pTrxFus without the amino-terminal hydrophobic region and expressed in Escherichia coli. The recombinant protein, purified essentially to homogeneity by affinity chromatography, catalyzes the sequential condensation of isopentenyl diphosphate with different allylic diphosphates, farnesyl diphosphate being the best allylic substrate of the reaction. All-trans-polyprenyl diphosphates containing 3-13 isoprene units are synthesized, which identifies the B318L protein as a trans-prenyltransferase.

African Swine Fever Virus↗

PubMed1997

Characterization of the African swine fever virus structural protein p14.5: a DNA binding protein.

The gene encoding the structural protein p14.5 of African swine fever virus (ASFV) has been mapped and sequenced. This gene, designated E120R, is located in the Sa/l H/EcoRl E restriction fragment of the ASFV genome and is predicted to encode a protein of 120 amino acids with a molecular weight of 13.4 kDa. Northern-blot analysis showed that E120R is transcribed at late times during the viral replication cycle. The E120R gene product has been expressed in Escherichia coli, purified, and used as an antigen for antibody production. The antiserum anti-pE120R recognized a protein in infected cell extracts with an apparent molecular mass of 14.5 kDa, named p14.5. This antiserum also detected protein p14.5 in purified virus particles. Protein p14.5 is synthesized late in infection and is located in viral factories. Immunoprecipitation analysis and binding-assay experiments have shown that protein p14.5 interacts with a protein that could correspond to the major structural protein p72. Purified protein p14.5 interacts with DNA in a sequence-independent manner. It binds to both single-stranded and double-stranded DNA. A possible role of protein p14.5 in the encapsidation of ASFV DNA is suggested.

African Swine Fever Virus↗