Biomedical subjects

E Viñuela

Publications and source records attributed to E Viñuela.

At least 55 records · Page 3Linked to original sources

PubMed1993

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication.

African Swine Fever Virus↗

PubMed1993

High-level expression in Escherichia coli of the gene coding for the major structural protein (p72) of African swine fever virus.

The gene encoding the major structural protein (p72) of African swine fever virus (ASFV) has been expressed in Escherichia coli using a T7 RNA polymerase system. The use of a recombinant plasmid which contains the entire gene inserted between the T7 promoter and the transcription terminator of the expression vector allowed us to obtain a high expression level of the intact viral protein. This polypeptide, which appears in the insoluble fraction of the bacterial extracts, showed an intense reaction with the antibodies present in the sera of ASFV-infected animals, as demonstrated by Western blot and enzyme-linked immunosorbent assay. The recombinant protein was purified by size-exclusion high-performance liquid chromatography and used to develop a serological test of the disease.

African Swine Fever↗

PubMed1993

Polyprotein processing in African swine fever virus: a novel gene expression strategy for a DNA virus.

This report shows that African swine fever virus (ASFV)--a large DNA-containing virus--synthesizes a polyprotein to produce several of its structural proteins. By immunoprecipitation analysis, we have found that ASFV polyprotein is a 220 kDa myristoylated polypeptide (pp220) which, after proteolytic processing, gives rise to four major structural proteins: p150, p37, p34 and p14. Processing of the ASFV polyprotein takes place at the consensus sequence Gly-Gly-X and occurs through an ordered cascade of proteolytic cleavages. So far, polyprotein processing as a mechanism of gene expression had been found only in positive-strand RNA viruses and retroviruses. According to the results presented here, ASFV is the first example of a DNA virus that synthesizes a polyprotein as a strategy of gene expression.

African Swine Fever Virus↗

PubMed1993

African swine fever virus guanylyltransferase.

The gene coding for the guanylyltransferase of African swine fever virus has been identified and sequenced. The gene, designated NP868R, is located within fragments EcoRI N' and D of the virus genome (BA71V strain) and encodes a protein with a predicted molecular mass of 99.9 kDa that shares significant similarity with the large subunit of both vaccinia and Shope fibroma virus capping enzymes, with percentages of identity of 20.6 and 21.8%, respectively. A protein of 95 kDa was induced in Escherichia coli cells transformed with a recombinant plasmid carrying the NP868R gene. The E. coli expressed protein, as well as a protein of the same molecular weight present in African swine fever virus particles, form a covalent complex with GTP that can be reversed by pyrophosphate, two characteristic reactions of guanylyltransferases. An examination of the amino acid sequences of the African swine fever virus, poxvirus, and yeast guanylyltransferases has revealed a common motif around a lysine residue at the amino-terminal part of the proteins [Y(V, A)X2K(T, A)DG] which resembles the adenylylation site of DNA ligases (Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991). Proc. Natl. Acad. Sci. USA 88, 400-404). This lysine residue could be the guanylylation site in these enzymes.

African Swine Fever Virus↗

PubMed1993

Localization of the African swine fever virus attachment protein P12 in the virus particle by immunoelectron microscopy.

The African swine fever virus attachment protein p12 was localized in the virion by immunoelectron microscopy. Purified virus particles were incubated, before or after different treatments, with p12-specific monoclonal antibody 24BB7 and labeled with protein A-colloidal gold. Untreated virus particles showed labeling only in lateral protrusions that followed the external virus envelope. Mild treatment of African swine fever virions with the nonionic detergent octyl-glucoside or with ethanol onto the electron microscope grid resulted in a heavier and more homogeneous labeling of the virus particles. In contrast, the release of the external virus proteins by either octyl-glucoside or Nonidet-P40 and beta-mercaptoethanol generated a subviral fraction that was not labeled by 24BB7. Preembedding, labeling, and thin-sectioning experiments confirmed that the antigenic determinant recognized by 24BB7 was localized into the external region of the virus particle but required some disruption to make it more accessible. From these results we conclude that protein p12 is situated in a layer above the virus capsid with, at least, one epitope predominantly not exposed in the virion surface; this epitope may not be related to the virus ligand-cell receptor interaction.

African Swine Fever Virus↗

PubMed1993

African swine fever virus encodes a DNA ligase.

Sequencing of the EcoRI N' fragment of African swine fever virus (ASFV) DNA revealed an open reading frame encoding a protein similar to ATP-dependent DNA ligases. When the gene encoding this protein was expressed in Escherichia coli, a protein of the expected molecular mass was labeled in bacterial extracts upon incubation with [alpha-32P]ATP. The recombinant protein comigrated in SDS-PAGE with the putative viral DNA ligase detected in extracts of infected cells. We demonstrate that the recombinant protein is a DNA ligase by dissociation of the protein-[32P]AMP adduct with pyrophosphate and nicked DNA. The putatively adenylylated lysine in ASFV is surrounded by two arginine residues, instead of by two hydrophobic amino acids as in the other ATP-dependent DNA ligases. This might explain the high concentration of pyrophosphate necessary to revert the DNA ligase--AMP adduct in ASFV, 10- to 100-fold higher than that required for other DNA ligases. A comparison of the amino acid sequences reported for ATP-dependent DNA ligases disclosed three new amino acid motifs around the adenylylation site of these enzymes. ASFV DNA ligase has little similarity to the other enzymes at the ends of the molecule, but conserves the amino acid motifs of the central region.

Adenosine Triphosphate↗

PubMed1993

Characterization of two African swine fever virus 220-kDa proteins: a precursor of the major structural protein p150 and an oligomer of phosphoprotein p32.

Two kinds of unrelated African swine fever virus proteins of 220 kDa have been identified by means of two-dimensional gel electrophoresis and immunoprecipitation analysis. One species, named pp220 and identified as the precursor of the major structural protein p150, was found to be a moderately acidic protein (pl near 7) expressed after the replication of the viral DNA. The second species, a cluster of 220-kDa proteins with slightly different isoelectric points (pl ranging from 5 to 6), was found to be a homooligomeric complex formed by an early 32-kDa protein. This component was identified as the viral phosphoprotein p32, the most immunogenic early protein of African swine fever virus. A detailed characterization of its oligomeric structure is reported.

African Swine Fever Virus↗

PubMed1993

Virus-host interactions in African swine fever: the attachment to cellular receptors.

Biochemical and morphological techniques have shown that African swine fever virus (ASFV) enters susceptible cells by a mechanism of receptor-mediated endocytosis. The virus binds to a specific, saturable site in the cell and this interaction is required for a productive infection. A structural ASFV protein of 12kDa (p12) has been identified to be involved in the recognition of the cellular receptor, on the basis of the specific binding of the polypeptide to sensitive Vero cells. Protein p12 is externally located in the virus particle, forming disulfide-linked dimers with an apparent molecular mass of 17kDa. The gene has been mapped within the central region of the BA71V strain genome. Sequencing analysis has shown the existence of an open reading frame encoding a polypeptide of 61 amino acids characterized by the presence of a putative transmembrane domain, and a cysteine rich region in the C-terminal part which may be responsible for the dimerization of the protein. Transcripts of the p12 gene were only synthesized during the late phase of the infectious cycle. No posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation or fatty acid acylation, have been found. The comparison of the amino acid sequence of protein p12 from 11 different virus strains has revealed a high degree of conservation of the polypeptide.

African Swine Fever↗

PubMed1993

Consumption coagulopathy associated with shock in acute African swine fever.

We studied the evolution of shock using a comprehensive array of haematological tests in pigs infected with the highly virulent strain Malawi '83 (Lilongwe 20/1). A sudden onset of illness was observed between day 5 and 7 after inoculation with development of flush, episodes of epistaxis and melaena. Prior to these clinical signs, initiation of a consumption coagulopathy was demonstrated with loss of antithrombin III and plasminogen activity. Our findings indicate that during infection with this highly virulent strain the development of a consumption coagulopathy precedes and possibly contributes to shock, which results in haemorrhage and death.

Acute Disease↗

PubMed1993

Haemostatic abnormalities in African swine fever a comparison of two virus strains of different virulence (Dominican Republic '78 and Malta '78).

African swine fever (ASF) virus strains cause haemorrhage by producing a variety of defects, which vary in severity from strain to strain. To distinguish the main haemostatic defects leading to haemorrhage, two groups of pigs were infected with moderately virulent (Dominican Republic '78) and less virulent (Malta '78) ASF virus strains. Mortality rate and severity of clinical observations were greater in pigs infected with DR '78 virus compared with pigs infected with Malta '78 virus. The animals became febrile from day 3 to 4 onwards at a time when the viraemia was high (10(7) to 10(8) HAD50/ml). No difference was found during the period observed in their pattern of viraemia or pyrexia. Thrombocytopenia developed in both groups but with different kinetics, suggesting two different mechanisms of sequestration of platelets. When coagulation tests were performed, significant abnormalities were found, including evidence for disseminated intravascular coagulation. These abnormalities were much less pronounced in the group infected with Malta '78. Antithrombin III activity did not change significantly in either group. Decreased plasminogen activity was found in the early phase of disease in DR '78 infected pigs. These results indicate that when haemorrhage does occur in DR '78 infected pigs, it is a consequence of more pronounced degrees of haemostatic impairment probably due to a marked endothelial injury and/or generation of procoagulant activity.

African Swine Fever↗

PubMed1993

Structure and expression in E. coli of the gene coding for protein p10 of African swine fever virus.

The gene encoding protein p10, a structural protein of African swine fever (ASF) virus, has been mapped, sequenced and expressed in E. coli. Protein p10 was purified from dissociated virus by reverse-phase HPLC, and its NH2-terminal end identified by automated Edman degradation. To map the gene encoding protein p10, a mixture of 20-mer oligonucleotides based upon a part of the amino acid sequence was hybridized to cloned ASF virus restriction fragments. This allowed the localization of the gene in fragment Eco RI K of the ASF virus genome. The nucleotide sequence obtained from this region revealed an open reading frame encoding 78 amino acids, with a high content of Ser and Lys residues. Several of the Ser residues are found in Ser-rich regions, which are also found in some nucleic acid-binding proteins. The gene coding for protein p10 has been inserted in an expression vector which contains the promoter for T7 RNA polymerase. The recombinant plasmid was used to produce the ASF virus protein in E. coli. The bacterially produced p10 protein shows a strong DNA binding activity with similar affinity for both double-stranded and single-stranded DNA.

African Swine Fever Virus↗

PubMed1993

Nucleotide sequence of a nucleoside triphosphate phosphohydrolase gene from African swine fever virus.

A putative nucleoside triphosphate phosphohydrolase (NTPase) gene of African swine fever virus was identified by using a degenerate oligonucleotide probe derived from the nucleoside triphosphate binding motif, which is highly conserved among viral and cellular NTPases. The probe hybridized with fragments SalI E and EcoRI Q, which is entirely contained in the former one. Sequencing of this region revealed an open reading frame, designated Q706L, coding for a protein of 706 amino acids, with a calculated molecular weight of 80,283. The deduced amino acid sequence of this open reading frame has significant similarity with the putative helicase encoded by the killer plasmid pGKL2 of Kluyveromyces lactis as well as with the NTPase I of vaccinia virus and entomopoxvirus and a subunit of the early transcription factor of vaccinia and fowlpox virus. The protein encoded by this open reading frame contains the sequence features characteristic of helicases of the superfamily II. According to this, we propose the inclusion of the product of this ASF virus gene in this superfamily.

Acid Anhydride Hydrolases↗

PubMed1993

Mapping and sequence of the gene encoding the African swine fever virion protein of M(r) 11500.

The gene encoding the African swine fever virus protein of M(r) 11,500, present in the virus particle, has been mapped and sequenced in the genome of the Vero cell-adapted virus strain BA71V. A serum raised against virion proteins of M(r) 12,000 to 13,000 isolated from polyacrylamide gels was used to screen a plasmid expression library, containing viral DNA random fragments, that expresses viral polypeptides fused to beta-galactosidase. Using this method, we have identified and sequenced the open reading frame (ORF) A137R, which initiates at the right end of the EcoRI A restriction fragment and extends into the EcoRI F fragment. Expression of the protein in Escherichia coli has confirmed that ORF A137R encodes a protein with an M(r) of about 12,000. A specific serum was raised against the E. coli-expressed protein, and has been used to identify the protein encoded by the ORF, which is translated at late times of infection and incorporated into the virus particle. Immunofluorescence experiments have shown that the protein localizes in virus factories.

African Swine Fever Virus↗

PubMed1993

African swine fever virus thymidylate kinase gene: sequence and transcriptional mapping.

A putative thymidylate kinase gene of African swine fever virus has been identified at the left end of the SalI I' fragment of the virus genome. The gene, designated A240L, has the potential to encode a protein of 240 amino acids with an M(r) of 27,754 and is transcribed early after infection. Primer extension analysis indicates that transcription is initiated a short distance from the first ATG codon of open reading frame A240L. The deduced amino acid sequence of this open reading frame shows significant similarity with the human, yeast and vaccinia virus thymidylate kinases, the degree of identity being 23.7, 25 and 23.5%, respectively. The putative African swine fever virus thymidylate kinase sequence is essentially collinear with the other thymidylate kinase sequences, but contains a carboxy-terminal extension of 37 amino acids rich in glutamic and aspartic acids. The A240L protein conserves the ATP-binding and nucleotide/nucleoside-binding domains characteristic of thymidylate kinases.

African Swine Fever Virus↗

PubMed1993

Transcriptional mapping of a late gene coding for the p12 attachment protein of African swine fever virus.

The transcriptional characterization of the gene coding for the p12 attachment protein of the African swine fever virus is presented. The results obtained have been used to generate the first detailed transcriptional map of an African swine fever virus late gene. Novel experimental evidence indicating the existence of major differences between the mechanisms controlling the transcription of late genes in African swine fever virus and poxviruses is provided.

African Swine Fever Virus↗

PubMed1993

Sequence and characterization of the major early phosphoprotein p32 of African swine fever virus.

The gene encoding protein p32, the most abundant and immunogenic protein induced by African swine fever virus at early times of infection, has been mapped in the EcoRI C' fragment of the genome of the Vero cell-adapted virus strain BA71V. Sequencing analysis has shown the existence of an open reading frame, named C'204L, encoding 204 amino acids. The protein is phosphorylated in serine residues located in the 115 N-terminal amino acids and was phosphorylated when expressed in cells infected with a vaccinia virus recombinant. Protein p32 is not glycosylated in spite of the presence of two putative N-glycosylation sites in the deduced amino acid sequence of the polypeptide. Immunofluorescence experiments have shown that the protein is localized in the cytoplasm of infected cells and not in the plasma membrane. In addition, the protein has been found in the soluble fraction and not in microsomes from BA71V-infected Vero cells. Low levels of the protein have been detected in the medium from infected swine macrophages, which probably corresponds to nonspecific release of cytoplasmic proteins. The protein encoded by other virus isolates shows different electrophoretic mobilities, indicating variability of p32.

African Swine Fever Virus↗

PubMed1993

African swine fever virus encodes a CD2 homolog responsible for the adhesion of erythrocytes to infected cells.

We have identified an open reading frame, EP402R, within the EcoRI E' fragment of the African swine fever virus genome that encodes a polypeptide of 402 amino acid residues homologous to the adhesion receptor of T cells, CD2. Transcription of EP402R takes place during the late phase of virus replication. The disruption of EP402R, achieved through the replacement of a 354-bp-long fragment from within EP402R by the marker gene lacZ, does not affect the virus growth rate in vitro but abrogates the ability of the virus to induce the adsorption of pig erythrocytes to the surface of infected cells. This result demonstrates that the protein encoded by EP402R is directly involved in the hemadsorption phenomenon induced by the infection of susceptible cells with African swine fever virus.

African Swine Fever Virus↗

PubMed1993

Inhibition of African swine fever virus binding and infectivity by purified recombinant virus attachment protein p12.

The African swine fever virus protein p12, involved in virus attachment to the host cell, has an apparent molecular mass of 17 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We have also identified 12- and 10-kDa forms of the p12 protein in infected Vero cells and found that the mature 17-kDa protein is the only form present in virus particles. The p12 protein has been produced in large amounts in Spodoptera frugiperda insect cells infected with a recombinant baculovirus. A 17-kDa protein that possessed the biological properties of the viral protein was produced, since it bound to susceptible Vero cells and not to receptor-negative L cells, which do not support virus replication. The binding of the baculovirus-expressed protein p12 to Vero cells was specifically blocked by virus particles. In addition, the recombinant protein purified by immunoaffinity chromatography blocked the specific binding of virus particles to susceptible cells and prevented infection, demonstrating that the p12 protein mediates the attachment of virions to specific receptors and indicating that blocking the p12-mediated interaction between African swine fever virus and receptors in Vero cells can inhibit infection. However, although antibodies specific for protein p12 are induced in natural infections and in animals inoculated with inactivated virus or recombinant protein p12, these antisera did not inhibit virus binding to the host cell or neutralize virus infectivity.

African Swine Fever Virus↗