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Biomedical subjects

D A Brian

Publications and source records attributed to D A Brian.

At least 55 records · Page 3Linked to original sources
PubMed1988

The amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation.

cDNA clones mapping within the first 2601 bases of the 3' end of the porcine transmissible gastroenteritis corona-virus (TGEV) genome were sequenced by the method of Maxam and Gilbert and an open reading frame yielding a protein having properties of the matrix (M or E1) protein was identified. It is positioned at the 5' side of the nucleocapsid (N) gene from which it is separated by an intergenic stretch of 12 bases. The deduced M protein comprises 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has a net charge of +7 at neutral pH. Thirty-four percent of its amino acid sequence is homologous with the M protein of the bovine coronavirus (BCV), 32% with that of the mouse hepatitis coronavirus (MHV), and 19% with that of the avian infectious bronchitis coronavirus (IBV). Judging from alignment with the BCV, MHV, and IBV M proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with only 28 for BCV, 26 for MHV, and 21 for IBV. Eleven of the sixteen amino-terminal amino acids are hydrophobic and the positions of charged amino acids around this sequence suggest that the first 16 amino acids comprise a potentially cleavable signal peptide for membrane insertion. A similar sequence is not found in the M proteins of BCV, MHV, or IBV. When mRNA from infected cells, or RNA prepared by in vitro transcription of the reconstructed M gene, was translated in vitro in the presence of microsomes, the M protein became translocated and glycosylated. When a protein without the amino-terminal signal peptide was made by translating a truncated version of the M gene transcript, some translocation and glycosylation also occurred suggesting that the amino-terminal signal peptide on the TGEV M protein is not an absolute requirement for membrane translocation. Interestingly, the amino-terminal peptide did not appear to be cleaved during in vitro translation in the presence of microsomes suggesting that a step in virion assembly may be required for proper exposure of the cleavage site to the signal peptidase.

Amino Acid Sequence↗
PubMed1988

Temporal regulation of bovine coronavirus RNA synthesis.

The structure and synthesis of bovine coronavirus (BCV)-specific intracellular RNA were studied. A genome-size RNA and seven subgenomic RNAs with molecular weights of approximately 3.3 X 10(6), 3.1 X 10(6), 2.6 X 10(6), 1.1 X 10(6), 1.0 X 10(6), 0.8 X 10(6) and 0.6 X 10(6) were detected. Comparisons of BCV intracellular RNAs with those of mouse hepatitis virus (MHV) demonstrated the presence of an additional RNA for BCV, species 2a, of 3.1 X 10(6) daltons. BCV RNAs contain a nested-set structure similar to that of other coronaviruses. This nested-set structure would suggest that the new RNA has a capacity to encode a protein of approximately 430 amino acids. Kinetic studies demonstrated that the synthesis of subgenomic mRNAs and genomic RNA are differentially regulated. At 4 to 8 h post-infection (p.i.), subgenomic RNAs are synthesized at a maximal rate and represent greater than 90% of the total viral RNA synthesized, whereas genome-size RNA accounts for only 7%. Later in infection, at 70 to 72 h p.i., genome-size RNA is much more abundant and accounts for 88% of total RNA synthesized. Immunoprecipitations of [35S]methionine-pulse-labeled viral proteins demonstrated that viral protein synthesis occurs early in the infection, concurrent with the peak of viral subgenomic RNA synthesis. Western blot analysis suggests that these proteins are stable since the proteins are present at high level as late as 70 to 72 h p.i. The kinetics of production of virus particles coincides with the synthesis of genomic RNA. These studies thus indicate that there is a differential temporal regulation of the synthesis of genomic RNA and subgenomic mRNAs, and that the synthesis of genomic RNA is the rate-limiting step regulating the production of virus particles.

Adenocarcinoma↗
PubMed1988

Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application.

A pneumopathic strain of bovine viral diarrhea virus was grown in cell culture and purified. Genomic ribonucleic acid was extracted, polyadenylated at the 3' end, and copied into complementary DNA after oligo-dT priming. Complementary DNA was male double stranded and cloned into the pUC9 plasmid. Approximately 200 complementary DNA clones varying in length from 0.5 to 2.5 kilobases were obtained. Hybridization assays indicated that the sequences isolated were specific for bovine viral diarrhea virus and that at least 5.5 kilobases of bovine viral diarrhea virus genome was represented in the library of complementary DNA clones, the majority of which may have originated from the 3' end of the virus genome. One cloned complementary DNA sequence was used as a 32P-labelled hybridization probe for bovine viral diarrhea virus detection. The probe hybridized with all cytopathic and noncytopathic strains of bovine viral diarrhea virus tested and was 100 times more sensitive than infectivity assays for the detection of bovine viral diarrhea virus. Hybridization did not occur with nucleic acids from bovine coronavirus, bluetongue virus, bovine adenovirus or uninfected cell cultures. Native plasmid DNA sequences, labelled with 32P, did not hybridize with bovine viral diarrhea virus ribonucleic acid.

Animals↗
PubMed1987

Nucleotide sequence of the porcine transmissible gastroenteritis coronavirus matrix protein gene.

cDNA clones mapping within the first 2601 bases of the 3' end of the TGEV genome were sequenced completely or in part by the method of Maxam and Gilbert and open reading frames were examined. One reading frame yielding a protein having properties of the matrix (M) protein was identified. It is positioned at the immediate 5' side of the nucleocapsid (N) gene but is separated by an intergenic region of 12 bases. The deduced M protein is comprised of 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has an amino acid sequence homology of approximately 36% with the mouse hepatitis coronavirus, 37% with the bovine enteric coronavirus, and 28% with the avian infectious bronchitis virus. Judging from an alignment with MHV and IBV proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with 26 for MHV and 21 for IBV.

Amino Acid Sequence↗
PubMed1987

Deduced amino acid sequence and potential O-glycosylation sites for the bovine coronavirus matrix protein.

The nucleotide sequence of the matrix (M) protein gene of the bovine coronavirus (BCV) was determined by sequencing cDNA clones derived from genomic RNA. The gene was found to map at the 5' side of the nucleocapsid protein gene and its sequence predicts a protein of 230 amino acids having a molecular weight of 26,376. The BCV M protein shares extensive sequence homology with the matrix protein of the mouse hepatitis coronavirus (MHV) but differs notably in the amino terminal region external to the virion envelope where BCV apparently uses at least two of its six potential O-glycosylation sites.

Amino Acid Sequence↗
PubMed1987

Glycosylation of the bovine coronavirus hemagglutinin protein.

The bovine coronavirus hemagglutinin protein gp140 is composed of disulfide-linked subunits of 65 kDa. This protein was further characterized with regard to its glycosylation. The glycosylated subunits of the protein are polypeptides having a molecular mass of 42.5 kDa. Both subunits appear to be processed to the same extent by the addition of N-linked oligosaccharides. Each subunit on the mature virion has 6-7 high mannose and 3-4 complex type carbohydrate chains attached to it.

Acetylglucosaminidase↗
PubMed1987

Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes.

The 3' end of the 20-kb genome of the Mebus strain of bovine enteric coronavirus (BCV) was copied into cDNA and cloned into the PstI site of the pUC9 vector. Four clones from the 3' end of the genome were sequenced either completely or in part to determine the sequence of the first 2451 bases. Within this sequence were identified, in order, a 3'-noncoding region of 291 bases, the gene for a 448-amino acid nucleocapsid protein (N) having a molecular weight of 49,379, and the gene for a 230-amino acid matrix protein (M) having a molecular weight of 26,376. A third large open reading frame is contained entirely within the N gene sequence but is positioned in a different reading frame; it potentially encodes a polypeptide of 207 amino acids having a molecular weight of 23,057. A higher degree of amino acid sequence homology was found between the M proteins of BCV and MHV (87%) than between the N proteins (70%). For the M proteins of BCV and MHV, notable differences were found at the amino terminus, the most probable site of O-glycosylation, where the sequence is N-Met-Ser-Ser-Val-Thr-Thr for BCV and N-Met-Ser-Ser-Thr-Thr for MHV. BCV apparently uses two of its six potential O-glycosylation sites.

Amino Acid Sequence↗
PubMed1987

Diagnosis of porcine and bovine enteric coronavirus infections using cloned cDNA probes.

Molecular clones representing the first 2,000 bases from the 3' end of the porcine transmissible gastroenteritis coronavirus genome and the first 2,160 bases from the 3' end of the bovine enteric coronavirus genome were used in dot blot hybridization assays to detect viral RNA from cell culture and from fecal specimens. In each case, the cloned DNA represents approximately 10% of the genome. The cloned sequence for each virus encompasses the 3' noncoding region, the nucleocapsid protein gene, and a large portion of the matrix protein gene. 32P-labeled cDNA probes prepared from these clones detected as little as 25 pg of RNA from the parental virus but did not detect RNA from the nonparental virus even when amounts of up to 10 ng per dot were used. This specificity reflects the antigenic diversity between these two coronaviruses. The hybridization assay could also detect coronaviruses antigenically closely related to the parental virus but not coronaviruses belonging to an antigenically unrelated subgroup. Dot blot hybridization for transmissible gastroenteritis coronavirus diagnosis was compared with the routine procedures of virus isolation and electron microscopy as a diagnostic test.

Animals↗
PubMed1986

Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene.

The 3' end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into cDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential, nonoverlapping genes were identified within the 3'-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3'-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.

Amino Acid Sequence↗
PubMed1986

Structural proteins of human respiratory coronavirus OC43.

The human respiratory coronavirus OC43 was grown on a human rectal tumor cell line and was isotopically labeled with amino acids, glucosamine, and orthophosphate to analyze virion structural proteins. Four major protein species were resolved by electrophoresis and many of their properties were deduced from digestion studies using proteolytic enzymes. The four proteins are: A 190 kDa protein, the presumed peplomeric protein, that was glycosylated and proteolytically cleavable by trypsin into subunits of 110 and 90 kDa. The subunits each represent a different amino acid sequence on the basis of peptide mapping; a 130 kDa protein that was glycosylated and behaved as a disulfide-linked dimer of 65 kDa molecules. It is the apparent virion hemagglutinin on the basis of digestion studies with trypsin, bromelain and pronase; a 55 kDa nucleocapsid protein that was phosphorylated; a 26 kDa matrix protein that was glycosylated. The 190, 130, 55 and 26 kDa species can therefore be designated P, H, N and M, respectively. They exist in molar ratios of 4:1:33:33, and are calculated to be present at the rate of 88, 22, 726, and 726 molecules per virion, respectively.

Cell Line↗
PubMed1985

Oligonucleotide fingerprints of antigenically related bovine coronavirus and human coronavirus OC43.

Virion RNAs from the bovine enteric coronavirus and the human respiratory coronavirus OC43 were compared by one dimensional gel electrophoresis and by oligonucleotide fingerprinting. For each virus, approximately 55 per cent of the RNA migrated as a 6.8 Md species, 10 per cent as a 0.68 Md species, and 15 per cent as heterogeneous small molecular weight RNA. A sequence homology of greater than 96 per cent was observed between the 6.8 Md species from the two viruses. The 0.68 Md RNA is apparently an intravirion, subgenomic, polyadenylated molecule based on RNAse studies, oligo (dT)-cellulose chromatography, and hybridization to a cDNA clone of the 3' terminal 1.19 Kb region of the bovine coronavirus genome.

Animals↗
PubMed1985

Bovine coronavirus hemagglutinin protein.

Treatment of purified bovine coronavirus (Mebus strain) with pronase destroyed the integrity of virion surface glycoproteins gp140, gp120, gp100, reduced the amount of gp26 and destroyed the hemagglutinating activity of the virus. Bromelain, on the other hand, destroyed the integrity of gp120, gp100 and gp26 but failed to remove gp140 and failed to destroy viral hemagglutinating activity. These experiments suggest that gp140 is the virion hemagglutinin. Immunoblotting studies using monospecific antiserum demonstrate that gp140 is a disulfide-linked dimeric structure reducible to monomers of 65 kDa.

Animals↗
PubMed1984

Antigenic relationships among proteins of bovine coronavirus, human respiratory coronavirus OC43, and mouse hepatitis coronavirus A59.

Antisera prepared against each of three single and one pair of major structural proteins of the bovine coronavirus (Mebus strain) were used in immunoblotting studies to measure cross-reactivity with the structural proteins of the human coronavirus OC43 and the mouse hepatitis coronavirus A59. We conclude that the bovine coronavirus is comprised of four major structural proteins, gp190 (normally present as 120- and 100-kilodalton subunits), gp140, pp52, and gp26. The human coronavirus OC43 has an antigenically homologous counterpart of similar molecular mass to each of these proteins. The mouse hepatitis coronavirus A59 has an antigenically homologous counterpart to only three of these proteins: gp190, pp52 and gp26. There is no counterpart in the mouse virus to the 140-kilodalton glycoprotein, the apparent hemagglutinin of the bovine coronavirus.

Animals↗
PubMed1982

RNA-dependent RNA polymerase activity in coronavirus- infected cells.

An enzymatic activity which incorporates [3H]UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with the mitochondrial fraction of infected cells, suggesting that the enzyme is membrane bound. The activity required the presence of all three ribonucleoside triphosphates in addition to [3H]UTP, and it was not inhibited by actinomycin D. The heated product was digested by RNase but not by DNase. Mg2+ was required for enzymatic activity, and its optimal concentration was approximately 5 mM. The size of the in vitro products was compared by electrophoresis with that of in vivo-synthesized virus-specified RNA to confirm the viral specificity of the polymerase activity. Virus-specified RNA from infected cells consisted of 10 species of single-stranded, polyadenylated RNA with molecular weights of 6.8 X 10(6), 6.2 X 10(6), 3.15 X 10(6), 1.40 X 10(6), 1.05 X 10(6), 0.94 X 10(6), 0.66 X 10(6), 0.39 X 10(6), 0.34 X 10(6), and 0.24 X 10(6). In vitro synthesized RNA consisted of a high-molecular-weight species, of apparently higher molecular weight than genomic RNA, and two single-stranded species that electrophoretically comigrated with the species of 1.40 X 10(6) and 0.66 X 10(6) molecular weight made in vivo.

Animals↗
PubMed1982

Bovine coronavirus structural proteins.

The tissue culture-adapted strain (Mebus) of bovine coronavirus was grown in the presence of isotopically labeled amino acids, glucosamine, or orthophosphate for the purpose of analyzing the virion structural proteins. Five species of polypeptides were identified when purified virions were solubilized in urea and sodium dodecyl sulfate and resolved by polyacrylamide gel electrophoresis. Four species were glycosylated and had apparent molecular weights of 140,000, 120,000, 100,000, and 26,000. The glycoproteins were susceptible to proteolytic cleavage and enzymatic iodination when intact virions were studied and are thus at least partially external to the virion envelope. The 140,000-molecular-weight glycoprotein is apparently a dimer of 65,000-molecular-weight glycopolypeptides held together by disulfide linkages. Species 5 was phosphorylated and had an apparent molecular weight of 52,000. In the intact virion, it was unaffected by protease and was not enzymatically iodinated. It is therefore apparently an internal protein.

Coronaviridae↗