Biomedical subjects

E Viñuela

Publications and source records attributed to E Viñuela.

At least 37 records · Page 2Linked to original sources

PubMed1997

Inhibition of apoptosis by the African swine fever virus Bcl-2 homologue: role of the BH1 domain.

The function of the African swine fever virus (ASFV) bcl-2 homologue, gene A179L, in the regulation of apoptosis was investigated using as a model system the human myeloid leukemia cell line K562 induced to die by apoptosis with inhibitors of macromolecular synthesis, a process that is prevented by overexpression of human bcl-2. It is shown that transfection of K562 cells with the ASFV A179L gene protects these cells from apoptotic cell death induced by a combination of cycloheximide and actinomycin D or by treatment with cytosine arabinoside. To test the functional role of the highly conserved BH1 domain present in the A179L protein, the Gly residue at position 85 was mutated to Ala, since it has been shown that substitution of the corresponding Gly in human Bcl-2 abrogates its death-repressor activity. It was found that the Gly-to-Ala mutation in the BH1 domain of the viral protein abolished its capacity to protect the K562 cells from apoptosis, indicating that this Gly is essential for A179L action. This finding stresses the functional similarity of the BH1 domains of the viral protein and cellular Bcl-2.

African Swine Fever Virus↗

PubMed1997

Protein cell receptors mediate the saturable interaction of African swine fever virus attachment protein p12 with the surface of permissive cells.

Previous studies have demonstrated that the entry of African swine fever virus (ASFV) into Vero cells and swine macrophages is mediated by saturable binding sites located on the plasma membrane. The ASFV protein p12 has been implicated in virus attachment to the host cell, but the cellular component responsible for the interaction with the virus is largely unknown. We have studied the binding of recombinant p12 and ASFV to different cell lines. Permissive cells were able to bind p12 in saturable and nonsaturable interactions, as reported for ASFV. Experiments of binding recombinant p12 have been used for the initial characterization of the specific receptors on Vero cells. The treatment of cell surfaces with different enzymes and lectins resulted in the inhibition of the p12 binding activity by several proteases, but not by glycosidases or lipase, suggesting that the receptor is composed of protein, with no carbohydrates or lipids involved in the virus attachment to the cellular membrane. The recovery of receptor activity after pronase treatment was completed in 6 h in culture medium containing tunicamycin, and could not be restored in the presence of cycloheximide, confirming that synthesis of new proteins, but not glycosylation, was required for the recovery of the receptor activity. These data support the idea that membrane protein(s) on the surface of permissive cells act as receptors for ASFV and that this specific interaction is, at least, one necessary step in a productive virus infection.

African Swine Fever Virus↗

PubMed1997

African swine fever virus-specific cytotoxic T lymphocytes recognize the 32 kDa immediate early protein (vp32).

African swine fever (ASF) virus-specific cytotoxic T lymphocyte (CTL) activity has been studied in a model in which SLA inbred minipigs were experimentally infected with an attenuated isolate of the virus. The CTL assays were performed using alveolar macrophages as target cells. The specific lysis is mediated by purified CD8+ lymphocytes but not by CD4+ cells and can be blocked by incubation with anti-SLA class I monoclonal antibodies. The purified CD8+ population produced high levels of interferon-gamma after ASF virus stimulation. In an attempt to define the viral proteins recognized by CTL, target cells infected with a recombinant vaccinia virus (VV) expressing the ASF virus p32, an immediate early protein during ASF virus replication, were recognized and lysed by CTL. This assay may be useful for VV recombinant screening in order to identify other potential target ASF virus proteins.

African Swine Fever Virus↗

PubMed1997

Assembly of African swine fever virus: role of polyprotein pp220.

Polyprotein processing is a common strategy of gene expression in many positive-strand RNA viruses and retroviruses but not in DNA viruses. African swine fever virus (ASFV) is an exception because it encodes a polyprotein, named pp220, to produce several major components of the virus particle, proteins p150, p37, p34, and p14. In this study, we analyzed the assembly pathway of ASFV and the contribution of the polyprotein products to the virus structure. Electron microscopic studies revealed that virions assemble from membranous structures present in the viral factories. Viral membranes became polyhedral immature virions after capsid formation on their convex surface. Beneath the lipid envelope, two distinct domains appeared to assemble consecutively: first a thick protein layer that we refer to as core shell and then an electron-dense nucleoid, which was identified as the DNA-containing domain. Immunofluorescence studies showed that polyprotein pp220 is localized in the viral factories. At the electron microscopic level, antibodies to pp220 labeled all identifiable forms of the virus from the precursor viral membranes onward, thus indicating an early role of the polyprotein pp220 in ASFV assembly. The subviral localization of the polyprotein products, examined on purified virions, was found to be the core shell. In addition, quantitative studies showed that the polyprotein products are present in equimolar amounts in the virus particle and account for about one-fourth of its total protein content. Taken together, these results suggest that polyprotein pp220 may function as an internal protein scaffold which would mediate the interaction between the nucleoid and the outer layers similarly to the matrix proteins of other viruses.

African Swine Fever Virus↗

PubMed1997

Proteolytic processing in African swine fever virus: evidence for a new structural polyprotein, pp62.

We have identified an open reading frame (ORF), CP530R, within the EcoRI C' fragment of the African swine fever virus (ASFV) genome that encodes a polyprotein of 62 kDa (pp62). Antisera raised against different regions of ORF CP530R recognized a polypeptide of 62 kDa in ASFV-infected cells during the late phase of virus replication, after the onset of viral DNA synthesis. Pulse-chase experiments showed that polyprotein pp62 is posttranslationally processed to give rise to two proteins of 35 kDa (p35) and 15 kDa (p15). This proteolytic processing was found to take place at the consensus sequence Gly-Gly-X through an ordered cascade of proteolytic cleavages like that which also occurs with ASFV polyprotein pp220 (C. Simón-Mateo, G. Andrés, and E. Viñuela, EMBO J. 12:2977-2987, 1993). Immunofluorescence studies showed that polyprotein pp62 is localized in the viral factories. In addition, immunoprecipitation analysis of purified virus particles showed that mature products p35 and p15 are major structural proteins. According to these results, polyprotein processing represents an essential strategy for the maturation of ASFV structural proteins.

African Swine Fever Virus↗

PubMed1996

Intermediate class of mRNAs in African swine fever virus.

A transcriptional analysis of the African swine fever virus (ASFV) I226R and I243L genes is presented. Steady-state kinetics and transfection experiments showed the existence of a new temporal class of ASFV mRNAs transcribed from these genes, with the characteristics of the poxvirus intermediate transcripts. Transcription of the I226R gene gave rise to intermediate and late mRNAs that started from different sites, while the I243L gene produced early, intermediate, and late mRNAs, also starting from different sites. The presence of intermediate genes suggests a cascade model for the regulation of ASFV gene expression.

African Swine Fever Virus↗

PubMed1995

The African swine fever virus IAP homolog is a late structural polypeptide.

The analysis of the complete nucleotide sequence of the African swine fever virus genome has revealed the existence of a number of genes potentially capable of modifying the host's response to the virus infection. In this report, we describe the results of the characterization of the A224L gene that encodes a novel member of the family of apoptosis inhibitors known as IAP proteins. A224L is expressed during the late phase of the infectious cycle, and its polypeptide product is assembled into virus particles.

African Swine Fever Virus↗

PubMed1995

Production and purification of recombinant African swine fever virus attachment protein p12.

The conditions for cultivation of Spodoptera frugiperda (Sf9) insect cells for production of recombinant baculoviruses have been studied, to scale-up and improve the efficiency of the process for production of the African swine fever virus attachment protein p12 in the baculovirus expression system. It was shown that the total virus and recombinant protein production in insect cells infected with the Acp12 recombinant baculovirus were slightly dependent on cell density, but largely dependent on the serum concentration, in the case of suspended cells, but not in static monolayer cultures. The yield of recombinant protein p12 exceeded 50 mg per 1 of 2 x 10(9) cells, representing more than 10% of total cell proteins, a level > 20-fold higher than that observed with other eukaryotic expression systems. The presence of p12 in the cytoplasmic fraction of infected cells has allowed the purification of the protein by a simple two-step procedure of aqueous phase partition and octyl-glucoside solubilization. The recombinant protein p12 was able to inhibit, in a dose-dependent manner, the African swine fever virus production in swine macrophages infected with a number of different virus isolates, including attenuated, virulent, highly passaged on tissue culture, and non-haemadsorbing strains, indicating a fundamental role for p12 in the early interaction of the virus with the natural target cell receptors. However, pigs immunized with purified recombinant p12 did not develop protective immunity against African swine fever.

African Swine Fever Virus↗

PubMed1995

Vectors for the genetic manipulation of African swine fever virus.

Plasmid vectors designed to facilitate the genetic manipulation of African swine fever virus (ASFV) are described. Our results demonstrate that the beta-glucuronidase enzyme (GUS) can be used to follow gene expression in ASFV-infected cells. Infectious plaques formed by ASFV expressing GUS are visually detectable, thus providing a simple and highly sensitive method for the selection of ASFV recombinants. These and previous results have allowed us to construct two chimeric gene cassettes that constitute the basic tools for the generation of vectors to carry out the deletion of multiple target sequences from the ASFV genome. These cassettes, formed by: (a) a virus promoter; (b) the coding sequence of a reporter gene, either Lac Z or gusA; and (c) a strong signal for the 3' end formation of ASFV mRNAs, can be easily isolated by endonuclease restriction from their corresponding plasmid vectors. A general insertion/coexpression plasmid vector, pEPV2, has also been constructed. pEPV2 facilitates the insertion of foreign genes, together with the Lac Z reporter, into the thymidine kinase locus of ASFV. The functionality of pEPV2 has been tested by generating a recombinant ASFV expressing the luciferase gene. The vectors presented in this report constitute the first reported set of tools for the genetic manipulation of ASFV.

African Swine Fever Virus↗

PubMed1995

Analysis of the complete nucleotide sequence of African swine fever virus.

We present an analysis of the complete genome of African swine fever virus (ASFV) strain BA71V, including 80 kbp of novel sequence and 90 kbp previously reported by several authors. The viral DNA is 170,101 nucleotides long and contains 151 open reading frames. Structural and/or functional information is available on 113 viral proteins. ASFV encodes five multigene families, putative membrane and secreted proteins, and enzymes involved in nucleotide and nucleic acid metabolism (including DNA repair) and protein modification. Database comparisons have provided clues about genes that may modulate the virus-host interaction, thus, possibly controlling ASFV virulence and persistence. The virus possesses genes similar to CD2, IkappaB, C-type lectins, MyD116/gadd34/gamma, 34.5, bcl-2/bax, iap, NifS, and ERV1, which may allow a viral regulation of cell adhesion, apoptosis, and redox metabolism, as well as of the host immune response against ASFV infection. The proteins encoded by different ASFV isolates are highly similar, the most variable ones being those belonging to multigene families, some membrane proteins, and those containing tandem repeats. DNA sequence data confirm the intermediate characteristics of ASFV between poxviruses and iridoviruses, supporting the notion that ASFV belongs to an independent virus family.

African Swine Fever Virus↗

PubMed1995

Mapping and sequence of the gene encoding protein p17, a major African swine fever virus structural protein.

The gene encoding protein p17, a major structural protein of African swine fever virus, has been mapped and sequenced. Protein p17 was purified from dissociated virus by reverse-phase HPLC and the amino acid sequence of a peptide obtained after digestion of protein p17 with cyanogen bromide was determined by automated Edman degradation. To map the gene encoding protein p17, a mixture of 17-mer oligonucleotides based upon a part of the amino acid sequence was hybridized to cloned African swine fever virus DNA restriction fragments. This allowed the location of the gene in the fragment EcoRI D of the viral genome. The nucleotide sequence of a Sa/l/KpnI fragment revealed an open reading frame designated D117R encoding a protein of 117 amino acids with a deduced molecular mass of about 13,000 Da. A transcriptional analysis revealed that the p17 gene is expressed late in the viral infection cycle.

Africa↗

PubMed1995

A set of African swine fever virus tandem repeats shares similarities with SAR-like sequences.

A group of cross-hybridizing DNA segments contained within the EcoRI restriction fragments U', X and J of a Vero cell-adapted strain (BA71V) of African swine fever virus (ASFV) were mapped and sequenced. Analysis of the nucleotide sequence revealed the presence of a set of long internal repeated sequences composed of five types of tandemly repeat units of about 200 bp. These tandem repeats contain a G-rich core of 10-14 nucleotides surrounded by regions with a high A + T content distributed in oligo(dA).oligo(dT) tracts. Next to the repeated sequences we detected two related open reading frames that are members of a new multigene family (multigene family 300). Comparison of DNA sequences from several virus isolates indicated that this region undergoes frequent rearrangements leading to either duplications or deletions of the repeat units. These ASFV repeated sequences share similarities with chromosomal alpha satellite DNA, the scaffold-associated region and satellite III of Drosophila. Similar tandemly repeated sequences have not been described in other viruses.

African Swine Fever Virus↗

PubMed1995

The role of fibrinolysis in the pathogenesis of the haemorrhagic syndrome produced by virulent isolates of African swine fever virus.

The activity of several proteins involved in fibrinolysis and the morphological changes in the blood vessel walls of pigs infected with highly virulent (Malawi'83) and moderately virulent (Dominican Republic '78-DR'78) ASF virus isolates were determined. Pigs infected with the Malawi'83 virus developed an increased fibrinolytic activity due to high plasma levels of tissue-plasminogen activator (t-PA) of 71.3 +/- 22.8 IU/ml (mean +/- SD), which correlated well with an increased activation of interstitial capillary endothelial cells and high levels of 1150 +/- 73.6 nM of fibrin monomer in the circulation. Animals infected with DR'78 virus, in contrast, showed an inhibition of fibrinolysis in the late stages of disease with almost a 5-fold increase of plasminogen activator inhibitor (PAI) activity of 196.0 AU/ml. These results suggest that activation of the fibrinolytic system in pigs infected with the Malawi'83 virus is probably due to increased formation and deposition of fibrin in the circulation, contributing to an increased bleeding tendency and higher mortality. On the contrary, animals infected with DR'78 virus developed an inhibition of fibrinolysis and thus a reduction in bleeding.

Acute Disease↗

PubMed1994

Nucleotide sequence and variability of the inverted terminal repetitions of African swine fever virus DNA.

African Swine fever virus (ASFV) genome is a large (170-190 kb) double-stranded DNA molecule with structural features similar to those of Poxviruses. Prominent among those features are the presence of a hairpin loop structure at the end of the DNA molecule and terminal-inverted repeats (TIR). The TIRs have been previously demonstrated by electron microscopy and cross-hybridization of terminal restriction fragments. We have determined the sequence of both left and right DNA ends from the BA71V virus strain. The TIR is composed of 2134 bp and was identical at both genome ends. As much as 82% of the TIR sequence is made up of short (27-35 bp) sequences repeated within the TIR, in five different sets of repeats. The central portion of the TIR is occupied by a 34-bp sequence which is repeated in tandem 33 times. Length differences were found within TIRs of virus clones isolated from a single infected animal. The ASFV TIR resembles in structure the TIR of the related Poxviruses.

African Swine Fever Virus↗

PubMed1994

Swine-reconstituted SCID mice as a model for African swine fever virus infection.

Injection of swine peripheral blood mononuclear cells into mice with severe combined immunodeficiency (SCID), resulted in the stable long-term establishment of a functional swine immune system (SCID-sw). Swine immunoglobulins were present in the serum of SCID-sw mice and swine cells were detected in the blood as well as in lymph nodes and spleen using monoclonal antibodies raised against cell subpopulations. Swine lymphocytes from reconstituted SCID mice responded in vitro to specific antigens or mitogens. When SCID-sw mice were challenged with African swine fever (ASF) virus, ASF virus-infected cells were detected in blood and spleen, and antiviral antibodies and virus-specific T cells were generated.

African Swine Fever Virus↗

PubMed1994

Multigene families in African swine fever virus: family 505.

Sequencing of restriction fragment EcoRI A-SalI C of African swine fever virus has revealed the existence of a multigene family, designated family 505 because of the average number of amino acids in the proteins, composed of seven homologous and tandemly arranged genes. All the genes of family 505 are expressed during infection. Primer extension analysis showed that transcription is initiated a short distance (3 to 62 nucleotides) from the start codon of the corresponding open reading frame. The proteins of family 505 showed similarity to those of family 360 from African swine fever virus. In particular, a striking conservation of three regions at the amino terminus of the polypeptides was observed.

African Swine Fever Virus↗

PubMed1993

The DNA polymerase-encoding gene of African swine fever virus: sequence and transcriptional analysis.

The putative DNA polymerase-encoding gene of African swine fever virus has been sequenced. The gene, designated G1207R, is located in the central region of the viral genome, and encodes a protein of 1207 amino acids (aa) with a predicted M(r) of 139,835. The gene is transcribed at both early and late stages of infection into a 4.1-kb RNA. Transcription is initiated at tsp, 8 nucleotides (nt) upstream from the start codon. Open reading frame (ORF) G1207R contains four direct repeats in tandem close to the 3'-end. Each repeat consists of 12 nt, coding for the reiterated sequence, K/NPAG. The deduced aa sequence of G1207R shows significant similarity with DNA polymerases from cellular and viral origin, belonging to the alpha-like family of DNA polymerases. In particular, the G1207R protein presents a colinear arrangement of all the 3'-->5' exonuclease and polymerization highly conserved aa regions characteristic of this group of DNA-dependent DNA polymerases.

African Swine Fever Virus↗

PubMed1993

Two putative African swine fever virus helicases similar to yeast 'DEAH' pre-mRNA processing proteins and vaccinia virus ATPases D11L and D6R.

Two open reading frames (ORFs) of African swine fever virus (ASFV) encoding putative helicases have been sequenced. The two genes, termed D1133L and B962L, are located in the central region of the viral genome, but are separated by about 40 kb of DNA. Both genes are expressed late during ASFV infection of Vero cells, after replication of viral DNA has begun. Contiguous to D1133L, three other ORFs (D129L, D79L and D339L), encoding putative proteins of unknown function, have been sequenced. Proteins D1133L and B962L contain the amino acid motifs that characterize helicases of superfamily II. D1133L is most similar to a group of putative helicases which includes two proteins of vaccinia virus (D11L and D6R) involved in transcription of the viral genome, their homologues in other poxviruses, the protein encoded by ORF 4 of the yeast plasmids, pGKL2 and pSKL, and the previously identified ASFV protein, Q706L. B962L resembles a group of RNA-helicase-like proteins which includes three proteins of Saccharomyces cerevisiae involved in pre-mRNA splicing (PRP2, PRP16 and PRP22), Drosophila melanogaster KURZ and MLE, and vaccinia virus 18R.

Adenosine Triphosphatases↗