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D A Brian

Publications and source records attributed to D A Brian.

At least 37 records · Page 2Linked to original sources
PubMed1993

The Coronaviridae now comprises two genera, coronavirus and torovirus: report of the Coronaviridae Study Group.

At the April 1992, mid-term meeting of the International Committee on Taxonomy of Viruses (ICTV) a proposal from the Coronaviridae Study Group (CSG) to include the torovirus genus in the Coronaviridae was accepted. Following another proposal, the arterivirus genus was removed from the Togaviridae but not assigned to another family. The arteriviruses have some features in common with the Coronaviridae but also have major differences. After much debate, culminating in September 1992, it was decided that the CSG would not recommend inclusion of arterivirus in the Coronaviridae. It was agreed that (a) the nomenclature used for coronavirus genes, mRNAs and polypeptides (Cavanagh et al., 1990) should be used for toroviruses, (b) that the small (about 100 amino acids) membrane-associated protein, which is distinct from the integral membrane glycoprotein M, associated with virions of infectious bronchitis (Liu & Inglis, 1991) and transmissible gastroenteritis (Godet et al., 1992) coronaviruses would be referred to by the acronym sM (lower case 's') and (c) that 'pol' (polymerase) should be used as a working term for gene 1, which comprises open reading frames (ORFs) 1a and 1b in both genera of the Coronaviridae.

Arterivirus↗
PubMed1992

The 9-kDa hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated.

The open reading frame potentially encoding a 78 amino acid, 9101 Da hydrophobic protein (HP) and, mapping at the 3' end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome, was shown to be expressed during virus replication. The cloned HP gene was placed in a plasmid under control of the T7 RNA polymerase promoter and in vitro translation of transcripts generated in vitro yielded a 9.1-kDa protein that was immunoprecipitable with porcine hyperimmune anti-TGEV serum. Antiserum raised in rabbits against a 31 amino acid synthetic polypeptide that represented the central hydrophilic region of HP specifically immunoprecipitated HP from TGEV-infected cells. HP was further shown to become associated with microsomal membranes during synthesis in vitro and was found to be closely associated with the endoplasmic reticulum and cell surface membranes in infected cells. The intracellular location of HP suggests that it may play a role in the membrane association of replication complexes or in virion assembly.

Amino Acid Sequence↗
PubMed1992

The nucleocapsid protein gene of bovine coronavirus is bicistronic.

For animal RNA viruses that replicate through an RNA intermediate, reported examples of bicistronic mRNAs with overlapping open reading frames in which one cistron is contained entirely within another have been made only for those with negative-strand or double-stranded genomes. In this report, we demonstrate for the positive-strand bovine coronavirus that an overlapping open reading frame potentially encoding a 23-kDa protein (names the I [for internal open reading frame] protein) and lying entirely within the gene for the 49-kDa nucleocapsid phosphoprotein is expressed during virus replication from a single species of unedited mRNA. The I protein was specifically immunoprecipitated from virus-infected cells with an I-specific antipeptide serum and was shown to be membrane associated. Many features of I protein synthesis conform to the leaky ribosomal scanning model for regulation of translation. This, to our knowledge, is the first example of a bicistronic mRNA for a cytoplasmically replicating, positive-strand animal RNA virus in which one cistron entirely overlaps another.

Animals↗
PubMed1991

A PCR-enhanced method for determining the 5' end sequence of mRNAs.

A method was developed to amplify ligated cDNA copies of mRNA 5' ends before cloning and sequencing. The method facilitates the study of the 5' end sequence on many molecules of an individual mRNA species. It can also be used to study the 5' end sequence on other kinds of single-stranded RNA molecules.

Base Sequence↗
PubMed1991

Minus-strand copies of replicating coronavirus mRNAs contain antileaders.

The 5' leader sequence on mRNAs of the porcine transmissible gastroenteritis coronavirus was determined and found to be 90 nucleotides in length. An oligodeoxynucleotide with a sequence from within the leader was used as a probe in Northern analysis on RNA from infected cells, and an antileader (a minus-strand copy of the leader sequence) was shown to be present on all mRNA minus-strand species. RNase protection analysis showed the antileader to be approximately the same length as the leader. The kinetics of antileader appearance was the same as that for the appearance of minus-strand RNA species. This, along with a demonstration that viral mRNAs become packaged, gives further support to the idea that coronavirus mRNAs can undergo replication via subgenomic mRNA-length replicative intermediates, and that input mRNAs from infecting virions may serve as initial templates for their own replication. In this sense, then, coronaviruses behave in part like RNA viruses with segmented genomes.

Animals↗
PubMed1991

The 5' end of coronavirus minus-strand RNAs contains a short poly(U) tract.

A radiolabeled oligodeoxynucleotide primer that anneals near the common 5' end of bovine coronavirus minus-strand RNAs was extended with reverse transcriptase, and a major product suggesting poly(U) tracts of 8 to 20 nucleotides was found. The extended primer molecules were ligated head to tail, amplified by the polymerase chain reaction, cloned, and sequenced, and poly(U) tracts of 9 to 26 nucleotides were found. Poly(A) tails of 100 to 130 nucleotides on the 3' end of coronavirus plus-strand mRNAs and genome must, therefore, be generated by a mechanism that uses only a short poly(U) template. This pattern contrasts with that of other cytoplasmic, polyadenylated, plus-strand animal RNA viruses which utilize a full-length poly(U) template for poly(A) synthesis.

Animals↗
PubMed1991

Sequencing PCR DNA amplified directly from a bacterial colony.

We show that PCR product asymmetrically amplified directly from a bacterial colony can be sequenced to yield results as good as those obtained when purified template DNA is used for the PCR amplification step. With either template, greater than 300 nucleotides can be read from a typical sequencing reaction. Taq DNA polymerase was used for both the PCR amplification and sequencing reactions.

Base Sequence↗
PubMed1990

Deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site.

The sequence of the spike (also called peplomer or E2) protein gene of the Mebus strain of bovine coronavirus (BCV) was obtained from cDNA clones of genomic RNA. The gene sequence predicts a 150,825 mol wt apoprotein of 1363 amino acids having an N-terminal hydrophobic signal sequence of 17 amino acids, 19 potential N-linked glycosylation sites, a hydrophobic anchor sequence of approximately 17 amino acids near the C terminus, and a hydrophilic cysteine-rich C terminus of 35 amino acids. An internal Lys-Arg-Arg-Ser-Arg-Arg sequence predicts a protease cleavage site between amino acids 768 and 769 that would separate the S apoprotein into S1 and S2 segments of 85690 and 65153 mol wt, respectively. Amino terminal amino acid sequencing of the virion-derived gp 100 spike subunit confirmed the location of the predicted cleavage site, and established that gp 120 and gp 100 are the glycosylated virion forms of the S1 and S2 subunits, respectively. Sequence comparisons between BCV and the antigenically related mouse hepatitis coronavirus revealed more sequence divergence in the putative knob region of the spike protein (S1) than in the stem region (S2).

Amino Acid Sequence↗
PubMed1990

Sequence and expression analysis of potential nonstructural proteins of 4.9, 4.8, 12.7, and 9.5 kDa encoded between the spike and membrane protein genes of the bovine coronavirus.

The nucleotide sequence between the spike and membrane protein genes in the bovine coronavirus (BCV) genome was determined by sequencing cDNA clones of the genome, and open reading frames potentially encoding proteins of 4.9, 4.8, 12.7, and 9.5 kDa, in that order, were identified. The 4.9- and 4.8-kDa proteins appear to be vestiges of an 11-kDa protein for which a single nucleotide deletion event in the central part of the gene gave rise to a stop codon. The consensus CYAAAC sequence precedes the 4.9-, 12.7-, and 9.5-kDa ORFs and predicts that transcription will start from each of these sites. Northern analyses using sequence-specific probes and oligo(dT)-selected RNA demonstrated that the predicted transcripts are made, and that these correspond to mRNAs 4, 5, and 5-1. BCV mRNA 4 appears to be a counterpart to mouse hepatitis virus (MHV) mRNA 4 which, in the MHV JHM strain, encodes the putative 15.2-kDa nonstructural protein. BCV mRNAs 5 and 5-1 appear to be used for the synthesis of the 12.7- and 9.5-kDa proteins, respectively, which demonstrates a pattern of expression strikingly different from that utilized by MHV. MHV makes its homologs of the 12.7- and 9.5-kDa proteins from the single mRNA 5. In vitro translation analyses demonstrated that the BCV 9.5-kDa protein, unlike its MHV counterpart, is poorly made from downstream initiation of translation. Thus, from a comparison between BCV and MHV we find evolutionary evidence for the importance of the CYAAAC sequence in regulating coronavirus transcription.

Amino Acid Sequence↗
PubMed1990

Structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein.

The sequence of the hemagglutinin-esterase (HE) gene for the Mebus strain of bovine coronavirus was obtained from cDNA clones, and its deduced product is a 47,700-kilodalton apoprotein of 424 amino acids. Expression of the HE protein in vitro in the presence of microsomes revealed N-terminal signal peptide cleavage and C-terminal anchorage but not disulfide-linked dimerization. Dimerization was observed only after expression in vivo, during which HE was also transported to the cell surface.

Amino Acid Sequence↗
PubMed1990

Bovine coronavirus mRNA replication continues throughout persistent infection in cell culture.

The existence of viral mRNA replicons was demonstrated in cells infected with the bovine coronavirus by showing a minus-strand counterpart and a corresponding replicative intermediate for each subgenomic mRNA species. mRNA replication is thus a universal property of coronaviruses, since this is now the third coronavirus for which it has been demonstrated. During the acute phase of infection (first 48 h), minus and plus strands accumulated at the same rate initially, but maximal accumulation of minus strands peaked earlier than that for plus strands, indicating that minus- and plus-strand levels are differentially regulated. In addition, packaged (input) mRNAs appeared to serve as templates for their own early replication. mRNA replication continued throughout establishment and maintenance of persistent infection (studied for 120 days), which is consistent with our hypothesis that mRNA replication contributes mechanistically to virus persistence. A replication-defective (potentially interfering) species of RNA existed transiently (beginning at day 2 and ending before day 76 postinfection), but because of its transient nature it cannot be considered essential to the long-term maintenance of virus persistence.

Animals↗
PubMed1989

Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons.

The genome of the porcine transmissible gastroenteritis coronavirus is a plus-strand, polyadenylylated, infectious RNA molecule of approximately 20 kilobases. During virus replication, seven subgenomic mRNAs are generated by what is thought to be a leader-priming mechanism to form a 3'-coterminal nested set. By using radiolabeled, strand-specific, synthetic oligodeoxynucleotide probes in RNA blot hybridization analyses, we have found a minus-strand counterpart for the genome and for each subgenomic mRNA species in the cytoplasm of infected cells. Subgenomic minus strands were found to be components of double-stranded replicative forms and in numbers that surpass full-length antigenome. We propose that subgenomic mRNA replication, in addition to leader-primed transcription, is a significant mechanism of mRNA synthesis and that it functions to amplify mRNAs. It is a mechanism of amplification that has not been described for any other group of RNA viruses. Subgenomic replicons may also function in a manner similar to genomes of defective interfering viruses to lead to the establishment of persistent infections, a universal property of coronaviruses.

Animals↗
PubMed1989

Synthesis and processing of the bovine enteric coronavirus haemagglutinin protein.

The haemagglutinin molecule on the bovine enteric coronavirus has been identified as a glycoprotein of 140K composed of disulphide-linked subunits of 65K. In this study, we have shown the subunits to be identical by demonstrating an unambiguous amino-terminal amino acid sequence. The unglycosylated subunit was found to have an Mr of 42.5K and to undergo rapid disulphide linkage and glycosylation. Glycosylation was found to be of the asparagine-linked type and some of the oligosaccharides underwent processing to complex forms. Studies with inhibitors of glycosylation suggested that a processing of the haemagglutinin oligosaccharide takes place on the virion whilst it is in the Golgi apparatus. Each haemagglutinin subunit on the mature virion was estimated to possess six or seven carbohydrate chains of either the high-mannose or hybrid type, and three or four chains of the complex type.

Amino Acid Sequence↗