Biomedical subjects

E Viñuela

Publications and source records attributed to E Viñuela.

At least 73 records · Page 4Linked to original sources

PubMed1992

Genes homologous to ubiquitin-conjugating proteins and eukaryotic transcription factor SII in African swine fever virus.

The nucleotide sequence of the 6004-bp EcoRI I fragment of African swine fever virus DNA has been determined. Translation of the sequence revealed eight closely spaced open reading frames (ORFs), three of them reading rightward and five leftward. Northern blot hybridization analysis indicated that ORFs I73R and I78R were transcribed early in infection, whereas ORFs I177L, I196L, and I329L were expressed at late times. Transcripts for ORFs I215L, I226R, and I243L were detected at low levels in early RNA and at higher levels in late RNA. The intergenic regions between genes I73R/I329L and I78R/I215L were characterized by the presence of direct repeats in tandem. Direct repetitions were also found within ORF I196L. The protein encoded by ORF I329L contained a putative cleavable signal peptide and an internal transmembrane domain, and that encoded by ORF I177L had an amino-terminal hydrophobic region with the characteristics of a "start-stop" sequence. ORF I243L encoded a protein similar to the eukaryotic elongation factor SII. The protein encoded by ORF I215L was homologous to the family of ubiquitin-conjugating proteins.

African Swine Fever Virus↗

PubMed1992

Role of the host cell nucleus in the replication of African swine fever virus DNA.

An examination by autoradiography of African swine fever virus-infected alveolar macrophages pulse labeled with [3H]thymidine showed that, at early times of viral DNA replication, the grains were localized exclusively in the nucleus in 20% of the cells, while in 45% the label was found in the cytoplasm. In the remaining 35%, newly synthesized DNA was detected in both the nucleus and the cytoplasm. At later times, the percentage of cells with grains in the nucleus decreased considerably. Pulse-chase experiments indicated that the DNA synthesized in the nucleus is then transported to the cytoplasm. The presence of virus-specific DNA sequences in the nucleus was confirmed by in situ hybridization of infected macrophages. Similar hybridization experiments with African swine fever virus-infected VERO cells followed by confocal microscopy also indicated the existence of a nuclear stage in the localization of the viral DNA. These results suggest a mechanism for African swine fever virus DNA replication with an initial stage in the nucleus followed by a cytoplasmic phase. Specific nuclear forms associated with the hybridization signal have been observed in African swine fever virus-infected macrophages and VERO cells. The nuclear forms seen in macrophages are consistent with a mechanism for the egress of the viral DNA from the nucleus that involves initial budding at the nuclear membrane.

African Swine Fever Virus↗

PubMed1992

A gene homologous to topoisomerase II in African swine fever virus.

A putative topoisomerase II gene of African swine fever virus was mapped using a degenerate oligonucleotide probe derived from a region highly conserved in type II topoisomerases. The gene is located within EcoRI fragments P and H of the African swine fever virus genome. Sequencing of this region has revealed a long open reading frame, designated P1192R, encoding a protein of 1192 amino acids, with a predicted molecular weight of 135,543. Open reading frame P1192R is transcribed late after infection into a 4.6-kb RNA. The deduced amino acid sequence of this open reading frame shares significant similarity with topoisomerase II sequences from different sources, with percentages of identity between 23 and 29%. The evolutionary relationships among the topoisomerase II sequences of ASF virus, eukaryotes and prokaryotes were analyzed and a phylogenetic tree was established. The tree indicates that the ASF virus topoisomerase II gene was present in the virus genome before protozoa, yeasts, and metazoa diverged.

African Swine Fever Virus↗

PubMed1992

Genetic manipulation of African swine fever virus: construction of recombinant viruses expressing the beta-galactosidase gene.

Homologous recombination is shown to be specifically induced in Vero cells by infection with African swine fever (ASF) virus. The frequency of recombination induced by ASF virus infection between cotransfecting plasmids is comparable to that found after infection with the prototype poxvirus, vaccinia virus. The induction of recombination is accompanied by replication of the plasmid templates in the ASF virus-infected cells. An ASF virus insertion/expression plasmid vector containing the Escherichia coli reporter gene beta-galactosidase (beta-gal) fused to a viral promoter sequence was constructed. Recombination between homologous sequences present in both the plasmid vector and the virus genome led to the generation of recombinant viruses expressing the beta-gal gene. Visual screening of beta-gal+ plaques allowed the isolation and plaque purification of recombinant ASF viruses. The characterization of a beta-gal+ virus isolate showed that the beta-gal gene had been stably inserted into the thymidine kinase locus of the virus genome, thus demonstrating that controlled genetic manipulation of ASF virus can be achieved by homologous recombination in infected cells.

African Swine Fever Virus↗

PubMed1992

An erythroid species-specific antigen of swine detected by a monoclonal antibody.

A monoclonal antibody (1AC11) has been produced which recognized the glycophorin of swine red blood cells. 1AC11 was specific for swine membrane erythrocytes. No other swine cells (leukocytes, macrophages, kidney and testis cells) nor red blood cells from all the tested mammalian species (goat, human, sheep, cattle, horse, rabbit, cat and guinea pig) were recognized. There was no blood group activity detected. Immunocytochemical analysis of blood vessel in the swine pituitary tissue showed that besides membrane erythrocytes, cytoplasmic molecules were recognized in some cells. Immunoblot analysis of both membrane and aqueous phase of chloroform/methanol fractions from swine erythrocytes showed that the monoclonal antibody 1AC11 reacts with the major sialoglycoprotein of apparent molecular weight 45,000 daltons.

Animals↗

PubMed1992

Interferon-gamma production by African swine fever virus-specific lymphocytes.

Peripheral blood mononuclear cells (PBMC) from inbred pigs that were immunized with autologous macrophages infected with the African swine fever (ASF) virus BA71V, a nonvirulent virus isolate, proliferated and produced interleukin-2 in response to homologous and heterologous isolates of the ASF virus. They produced, however, interferon (IFN) only when challenged in vitro with homologous or attenuated isolates of the ASF virus, but not with heterologous or virulent isolates. The IFN was pH 2 labile and was neutralized by specific serum to porcine recombinant IFN gamma.

African Swine Fever↗

PubMed1992

Transcriptional analysis of multigene family 110 of African swine fever virus.

A transcriptional analysis of the 3.2-kb region of the African swine fever virus genome containing the five members of the multigene family 110 is presented. The mRNAs corresponding to the genes studied have short leader sequences with no intervening AUG codons before the translational start site, and their 3' ends map within a conserved sequence motif formed by a stretch of seven or more consecutive thymidylate residues. The possible role of this sequence as a signal for the 3'-end formation of African swine fever virus mRNAs is discussed. While four of the genes studied are actively transcribed from the beginning of the infection until the onset of virus DNA replication, the transcription of one of the members of the multigene family 110, the L270 gene, is silenced at an earlier time. A detailed analysis, including in vitro translation of mRNAs isolated from infected Vero cells, revealed that the L270 gene belongs to a small subset of early genes, designated immediate early, whose transcription is silenced before the onset of virus DNA replication. The transcriptional data obtained enabled us to generate the first detailed transcriptional map of a region of the African swine fever virus genome, thus opening the possibility of studying the cis-acting sequences involved in transcriptional control of the viral genes.

African Swine Fever Virus↗

PubMed1992

Amino acid sequence and structural properties of protein p12, an African swine fever virus attachment protein.

The gene encoding the African swine fever virus protein p12, which is involved in virus attachment to the host cell, has been mapped and sequenced in the genome of the Vero-adapted virus strain BA71V. The determination of the N-terminal amino acid sequence and the hybridization of oligonucleotide probes derived from this sequence to cloned restriction fragments allowed the mapping of the gene in fragment EcoRI-O, located in the central region of the viral genome. The DNA sequence of an EcoRI-XbaI fragment showed an open reading frame which is predicted to encode a polypeptide of 61 amino acids. The expression of this open reading frame in rabbit reticulocyte lysates and in Escherichia coli gave rise to a 12-kDa polypeptide that was immunoprecipitated with a monoclonal antibody specific for protein p12. The hydrophilicity profile indicated the existence of a stretch of 22 hydrophobic residues in the central part that may anchor the protein in the virus envelope. Three forms of the protein with apparent molecular masses of 17, 12, and 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been observed, depending on the presence of 2-mercaptoethanol and alkylation with 4-vinylpyridine, indicating that disulfide bonds are responsible for the multimerization of the protein. This result was in agreement with the existence of a cysteine-rich domain in the C-terminal region of the predicted amino acid sequence. The protein was synthesized at late times of infection, and no posttranslational modifications such as glycosylation, phosphorylation, or fatty acid acylation were detected.

African Swine Fever Virus↗

PubMed1992

Comparison of the sequence of the gene encoding African swine fever virus attachment protein p12 from field virus isolates and viruses passaged in tissue culture.

Comparison of the amino acid sequence of the African swine fever virus attachment protein p12 from different field virus isolates, deduced from the nucleotide sequence of the gene, revealed a high degree of conservation. No mutations were found after adaptation to Vero cells, and a polypeptide with similar characteristics was present in an IBRS2-adapted virus. The sequence of the 5' flanking region was conserved among the isolates, whereas sequences downstream of the gene were highly variable in length and contained direct repeats in tandem that may account for the deletions found in different isolates. Protein p12 was synthesized in swine macrophages infected with all of the viruses tested.

African Swine Fever↗

PubMed1991

Escherichia coli thymidine kinase: nucleotide sequence of the gene and relationships to other thymidine kinases.

The thymidine kinase (TK)-encoding gene (tdk) of Escherichia coli is located at min 27 of the E. coli genetic map. Sequence analysis of this region revealed an open reading frame of 205 codons. Identification of this region as the E. coli tdk gene was confirmed by its similarity to other TK-encoding genes. The E. coli amino acid (aa) sequence showed significant similarity to the corresponding TK polypeptides of vertebrates and large DNA viruses, but showed no similarity to known herpes virus TK enzymes. Mapping of highly conserved positions among all sequences indicates the importance of these residues for catalytic activity and may facilitate further functional studies. Using a distance matrix method, the evolutionary relationships among the TK aa sequence of poxviruses, eukaryotes and prokaryotes were analyzed and a potential phylogenetic tree was established.

Amino Acid Sequence↗

PubMed1991

Protein p22 of African swine fever virus: an early structural protein that is incorporated into the membrane of infected cells.

The open reading frame K'177, located at the left end of the African swine fever virus genome, codes for an early induced structural protein of 22,000 Da (p22), which is released from the viral particle by the nonionic detergent n-octyl-beta-D-glucopyranoside under conditions that solubilize external viral structural proteins. The predicted amino acid sequence of the protein contains a hydrophobic region at its N-terminus with characteristics of a signal peptide and, at early times after virus infection of Vero cells, the protein can be detected in the cell membrane by immunolabeling.

Amino Acid Sequence↗

PubMed1991

African swine fever virus fatty acid acylated proteins.

Labeling experiments with [3H]palmitic and [3H]myristic acids of African swine fever virus-infected Vero cells have shown that 11 proteins induced during infection are covalently bound to myristic acid and that palmitic acid was not attached to viral proteins. The time course of synthesis of the myristylated polypeptides and the requirements of viral DNA replication indicated that the myristylated proteins, with the exception of a 13-kDa protein, belong to the late class of viral proteins. The myristic moiety was not released by hydroxylamine treatment, suggesting that the fatty acid is bound to the polypeptide chain through an amide linkage. The purification of [3H]myristic acid-labeled extracellular virus particles demonstrated that the myristylated 28- and 13-kDa proteins incorporated into the virion.

Acylation↗

PubMed1991

The sequences of the ribonucleotide reductase genes from African swine fever virus show considerable homology with those of the orthopoxvirus, vaccinia virus.

Two African swine fever virus (ASFV) recombinant plasmids containing large inserts of DNA have been sequenced at random, and translations of the DNA sequence have been compared to libraries of vaccinia virus protein sequences. Among other genes identified by their extensive homology with vaccinia virus genes were the large and small subunits of ribonucleotide reductase. A 5.5-kb fragment from the Malawi (LIL20/1) strain of ASFV was identified as containing the genes for both these subunits. The fragment has been sequenced and the two genes have been found to be in a head-to-head orientation. The sequences are compared to other sequenced ribonucleotide reductase genes, and the evolutionary implications discussed.

African Swine Fever Virus↗

PubMed1991

Fc receptors do not mediate African swine fever virus replication in macrophages.

Titration experiments in swine macrophages have shown that African swine fever virus infectivity was not enhanced in the presence of antiviral antibodies. The early viral protein synthesis and the viral DNA replication in swine macrophages infected with virus-antibody complexes were inhibited in the presence of high doses of uv-inactivated virus, which saturated specific virus receptors, but not when Fc receptors were saturated with antibodies. These results indicate that African swine fever virus does not infect swine macrophages through Fc receptors and that the normal entry pathway through virus receptors is not bypassed by the virus-antibody complexes.

African Swine Fever Virus↗

PubMed1991

Isolation and molecular characterization of the swinepox virus thymidine kinase gene.

Swinepox virus (SPV), the only member of the Suipoxvirus genus, shows little antigenic relatedness or DNA homology to members of the other poxvirus genera. A SPV thymidine kinase (TK) gene was detected and mapped to the left end of the HindIII G fragment using degenerate oligonucleotide probes. Cloning and sequencing of a 1.8-kb HindIII-BamHI fragment containing the SPV TK gene revealed an open reading frame (ORF) of 181 amino acids yielding a predicted polypeptide of Mr 20.6 kDa with significant homology to both poxvirus and vertebrate thymidine kinases. Comparison with other TK protein sequences showed that the SPV thymidine kinase was closely related to the TK genes of avipoxviruses (52.0%) and vertebrates (57.1-59.7%). The TK gene from African swine fever virus (ASF) showed little homology (30.5%) to the SPV TK gene suggesting that these two viruses are not closely related though they share many biochemical features and infect a single, common mammalian host (swine). The SPV TK gene, like that of other poxviruses, is transcribed early, and when cloned into a TK- strain of vaccinia converted the virus to a TK+ phenotype. BUdRR mutants of SPV contained frameshift, deletion, and missense mutations in the TK ORF.

Amino Acid Sequence↗

PubMed1991

Repetitive nucleotide sequencing of a dispensable DNA segment in a clonal population of African swine fever virus.

Repetitive nucleotide sequencing of a dispensable genomic segment of a clonal population of African swine fever (ASF) virus has been carried out to estimate the mutant frequency to neutral alleles. Since no mutations have been detected in a total of 54026 nucleotides screened, the maximum mutant frequency is 5.5 x 10(-5) substitutions/nucleotide (95% confidence level). The result renders very unlikely the occurrence of hypermutational events during ASF virus DNA replication, at least within the selected DNA fragment.

African Swine Fever Virus↗

PubMed1991

Highly efficient expression of proteins encoded by recombinant vaccinia virus in lymphocytes.

Using a recombinant vaccinia virus (VV) that expresses E. coli beta galactosidase (beta-Gal) to infect lymphocytes, we show that enzymometrically or immunologically detectable beta-Gal expression is less pronounced among T cells than among B cells. VV infection caused growth inhibition of B cells, but barely affected T-cell proliferation in vitro. Moreover, the production of infectious viral particles was less pronounced in T lymphocytes. Kinetic studies revealed that after an initial dose-dependent growth inhibition, T cells continued to proliferate without the doubling time being affected by VV infection. Nonetheless, the T cells do express proteins encoded by recombinant VV, such as beta-Gal, or secrete soluble proteins such as interleukin-4, though at a lower efficiency at the per cell level than B lymphocytes. In conclusion, the physiology of T cells appears to be less perturbed by VV than that of B cells, although the virus is capable of directing expression of recombinant genes to T lymphocytes.

Animals↗

PubMed1991

African swine fever virus attachment protein.

Treatment of African swine fever virus particles with nonionic detergents released proteins p35, p17, p14, and p12 from the virion. Of these proteins, only p12 bound to virus-sensitive Vero cells but not to virus-resistant L or IBRS2 cells. The binding of p12 was abolished by whole African swine fever virus and not by similar concentrations of subviral particles that lacked the external proteins. A monoclonal antibody (24BB7) specific for p12 precipitated a protein that, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol, showed a molecular mass of 17 kDa (p17*) instead of 12 kDa as found in the presence of 2-mercaptoethanol. The relationship between these two proteins was confirmed by the conversion of p17* to p12 when the former was isolated from polyacrylamide gels in the absence of 2-mercaptoethanol and subsequently treated with the reducing agent. The supernatant obtained after immunoprecipitation with the p12-specific antibody lacked the virus-binding protein.

African Swine Fever Virus↗