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M S Collett

Publications and source records attributed to M S Collett.

At least 37 records · Page 2Linked to original sources
PubMed1991

Bovine viral diarrhea virus genomic organization.

In previous work, we developed a preliminary description of the genetic organization of the prototypic pestivirus bovine viral diarrhea virus (BVDV). In order to refine this genetic map and to further elucidate the gene products and expression strategy of this virus, we have generated a broad panel of sequence-specific antibody reagents. Use of these reagents not only allowed the identification of several previously undescribed viral polypeptides, but when used in in vivo pulse-chase experiments, they identified precursor polyproteins and processing intermediates. Data generated from these studies provide a more accurate and complete view of viral gene organization, as well as insight into several aspects of protein processing and the gene expression strategy employed by this pestivirus. These experiments also revealed varying stability and turnover rates for the mature BVDV proteins. These latter results have implications for the functional roles of certain gene products.

Animals↗
PubMed1991

Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing.

Bovine viral diarrhea virus (BVDV), the prototypic pestivirus, possesses a positive-strand RNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. We have endeavored to elucidate the mechanisms involved in protein biogenesis by this pestivirus. Here, we present our studies on gene expression from the viral nonstructural protein coding region encompassing the carboxy-terminal 60% of the ORF. Previous sequence and modeling analyses predicted the amino-terminal region of the BVDV nonstructural protein p80 to be a trypsin-like serine proteinase. Using a mammalian cell transient expression system, we show that this region indeed possessed a proteolytic activity and, further, required the serine residue previously predicted to be the putative serine proteinase catalytic site. We found the p80-region proteinase activity was required for proteolytic processing of all viral nonstructural proteins. Cleavage by this activity at the amino and carboxy termini of the p80 protein itself likely occurred intramolecularly (in cis), since we were unable to demonstrate activity in trans at these sites. Cleavages at the three processing sites downstream of the carboxy terminus of p80 were shown to occur in trans. However, p80 proteinase activity alone was not sufficient for cleavage of the last of these sites. Another viral gene product, or specific condition, is implicated as a necessary cofactor for p80 proteinase activity at this site. Pestivirus polyprotein processing can now be compared to similar events by viruses of other groups. Finally, the potential role of p80 proteinase activity in the phenotype of cytopathic biotypes of BVDV is discussed.

Animals↗
PubMed1991

Pestivirus gene expression: the first protein product of the bovine viral diarrhea virus large open reading frame, p20, possesses proteolytic activity.

The positive-strand RNA genome of pestiviruses contains a single large open reading frame (ORF) extending its entire length and is capable of encoding 450 kDa of protein. Studies have been undertaken with the purpose of elucidating the specific mechanisms involved in the biogenesis of the complete complement of pestivirus proteins. Here, we report on gene expression at the 5' end of the genome of the prototype pestivirus, bovine viral diarrhea virus (BVDV). We demonstrate, using both a cell-free transcription-translation system and a mammalian-cell transient-expression system, that the first protein product of the large ORF of BVDV, the p20 protein, possesses a specific proteolytic activity. The p20 proteinase activity acts to release the p20 protein from the nascent polyprotein. The p20 proteinase activity is not, however, required for downstream glycoprotein processing, indicating translocation of the pestivirus glycoprotein precursor is affected by an internal signal sequence.

Amino Acid Sequence↗
PubMed1990

Expression strategy of a phlebovirus: biogenesis of proteins from the Rift Valley fever virus M segment.

The middle (M) RNA segment of Rift Valley fever virus (RVFV) encodes four proteins: the major viral glycoproteins G2 and G1, a 14-kilodalton (kDa) protein, and a 78-kDa protein. These proteins are derived from a single large open reading frame (ORF) present in the virus-complementary M-segment mRNA. We used recombinant vaccinia viruses in which sequences representing the M-segment ORF were engineered as a surrogate system to study phlebovirus protein expression. To investigate the translational initiation codon requirements for synthesis of these proteins, we constructed a series of vaccinia virus recombinants containing specific sequence changes which eliminated select ATG codons found in the region of the ORF preceding the mature glycoprotein-coding sequences (the preglycoprotein region). Examination of phleboviral proteins synthesized in cells infected with these vaccinia virus recombinants clearly showed that the first ATG of the ORF was required for the production of the 78-kDa protein, while synthesis of the 14-kDa protein was absolutely dependent on the second in-phase ATG codon. Efficient biosynthesis of glycoprotein G2 was shown to depend on one or more ATG codons within the preglycoprotein region, but not the first one of the ORF. Synthesis of about one-half of the total glycoprotein G1 was affected by the amino acid changes that eliminated ATG codons, while production of the remainder appeared to be independent of all ATG codons in the preglycoprotein region. These data indicated that the means for glycoprotein G1 biosynthesis was distinct from those of the other three M-segment gene products. The results presented herein suggest that a surprisingly complex expression strategy is employed by the RVFV M segment. Although the full nature of the mechanisms involved in the biogenesis of the four RVFV M-segment proteins remains unclear, it does involve the use of at least two (ATG codons 1 and 2), and likely more, distinct translation start sites within the same ORF to produce its complete complement of gene products.

Base Sequence↗
PubMed1989

Baculovirus expression of the M genome segment of Rift Valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins.

Autographa californica nuclear polyhedrosis viral recombinants containing coding information for the Rift Valley fever virus (RVFV) envelope glycoproteins (G1 and G2) and varying amounts of preglycoprotein coding sequences were prepared by using transfer vectors pAc373 or pAcYM1. Expression products were processed to yield proteins indistinguishable from authentic G1 and G2 by gel electrophoresis. The immunogenic properties of the expressed proteins were assessed by immunizing mice and challenging with RVFV. A single inoculation with lysates of cells infected with recombinants expressing both G1 and G2 induced neutralizing antibody responses in mice and protected them from an otherwise lethal challenge with RVFV. Lysates of cells infected with a recombinant expressing only G2 also induced a protective response after two immunizations. Survivors displayed elevated antibody titers to G1 and G2 and also developed antibodies to the RVFV nucleocapsid protein, the latter allowing discrimination from vaccinated mice and indicating that animals had survived infection. Nonimmune mice were protected from lethal RVFV infection by passive transfer of sera from animals immunized with recombinant antigens, indicating that a humoral immune response is sufficient to protect against RVFV.

Animals↗
PubMed1989

Rift Valley fever virus M segment: phlebovirus expression strategy and protein glycosylation.

The M segment RNA of Rift Valley fever virus (RVFV) encodes four gene products: the two viral envelop glycoproteins G2 and G1, a glycosylated 78-kDa protein, and a nonglycosylated 14-kDa protein. These proteins are generated from a single open reading frame (ORF) by a strategy involving independent translational initiations at both the first and second in-phase ATG codons and co-translational processing of primary polyprotein products. The ORF encodes six sites for N-linked glycosylation: one present in the "preglycoprotein region" preceding the coding sequences of the mature envelop glycoproteins, and within the coding sequences of both the 78- and 14-kDa proteins; one site in the glycoprotein G2 coding region, also present in the 78-kDa protein; and four sites within glycoprotein G1. From analyses of RVFV proteins produced in cells infected with recombinant vaccinia viruses expressing various M segment regions, we show glycoprotein G2 was glycosylated at its single site and glycoprotein G1 at at least three sites. Both sites for N-linked glycosylation in the 78-kDa protein were occupied with glycan. This latter result indicated the preglycoprotein region glycosylation site was utilized in the 78-kDa protein, but this same site within the 14-kDa protein was not. Further analysis showed utilization of this glycosylation site, as well as proteolytic processing at the amino terminus of the mature glycoprotein G2, appeared to be determined by initiation codon usage. The two-site translational initiation expression strategy of this phlebovirus M segment and its role in the control of post-translational protein modification and processing are discussed.

Bunyaviridae↗
PubMed1988

CD15 monoclonal antibodies react with a phosphotyrosine-containing protein on the surface of human neutrophils.

mAb are useful as probes in the study of the roles of cell-surface components in neutrophil function. Many mAb that bind to human neutrophils react with the oligosaccharide lacto-N-fucopentaose III (CD15 antibodies). These antibodies, as well as several other widely used mAb reactive with human neutrophils, were employed to detect phosphoproteins present on these cells. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [gamma-32P]ATP revealed a 170 to 190-kDa phosphoprotein specifically reactive with CD15 antibodies. No phosphoproteins were immunoprecipitated by CD11 or CD18 mAb. Phosphoamino acid analysis of the 170- to 190-kDa protein showed that it contained predominantly phosphotyrosine and a low level of phosphoserine. Recently, it was shown that this phosphoprotein is one of the major substrates of ecto-protein kinase activity on human neutrophils. The roles for the 170- to 190-kDa phosphoprotein and the ecto-protein kinase in neutrophil function remain to be determined.

Antibodies, Monoclonal↗
PubMed1988

Rift Valley fever virus M segment: cellular localization of M segment-encoded proteins.

The Phlebovirus Rift Valley fever virus (RVFV), like other members of the Bunyaviridae family, matures intracellularly at the smooth-surfaced vesicles in the Golgi region of infected cells. Here we show that in cultured cells the RVFV glycoproteins G2 and G1 accumulate and are retained at this site. To investigate the parameters governing this subcellular localization, we have engineered portions of the cloned RVFV M segment (which encodes a 14- and a 78-kDa protein, in addition to glycoproteins G2 and G1) into vaccinia virus. Immunofluorescent analysis of cells infected with a vaccinia virus recombinant containing the entire open reading frame of the RVFV M segment revealed Golgi localization for glycoproteins G2, G1, the 78-kDa protein, and Golgi as well as some reticular distribution for the 14-kDa protein. These distributions paralleled those seen in authentic RVFV-infected cells. RVFV-vaccinia virus recombinants possessing progressive deletions within the 152 amino acid preglycoprotein sequence of the M segment were analyzed for possible effects on the cellular distribution of G2 and G1. Removal of the first 130 amino acids of the open reading frame amino-terminal to the mature glycoprotein coding sequences, while abolishing production of the 78- and 14-kDa proteins, did not alter the Golgi location of G2 and G1. The data suggest that Golgi-specific signals reside within the G2 and/or G1 glycoprotein sequences. The use of vaccinia virus recombinants provides a genetically manipulable expression system with which to further investigate the sequences involved in the intracellular localization of these Phlebovirus proteins.

Animals↗
PubMed1988

Rift Valley fever virus M segment: cell-free transcription and translation of virus-complementary RNA.

A cell-free system has been used to study gene expression of the M segment RNA of the Phlebovirus Rift Valley fever virus (RVFV). RVFV sequence-containing plasmids were used to synthesize M segment mRNA-like transcripts. These transcripts were then translated in vitro in the absence or presence of microsomal membranes. Cell-free translation of a transcript which closely resembled authentic M segment mRNA (RNA-7) yielded a primary translation product of 133 kilodaltons (kDa), the size expected of a polypeptide encompassing the entire open reading frame (ORF) of the M segment. When translations were conducted in the presence of microsomal membranes, this primary protein was cotranslationally processed to yield the two viral glycoproteins, G1 and G2, as well as proteins of 78, 21, and 14 kDa. With one exception, these in vitro processed polypeptides comigrated with M segment-encoded proteins found in RVFV-infected cell lysates. A polypeptide corresponding to the in vitro 21-kDa protein was not detected in vivo. To investigate translational initiation and processing of the protein products of the M segment, additional transcripts were generated in which varying portions of the amino-terminal "preglycoprotein" region of the M segment ORF were deleted. Translation results indicated that the 78- and 21-kDa proteins were initiated from the first methionine codon of the ORF, and the 14-kDa polypeptide began from the second in-phase ATG. These products and a major portion of the preglycoprotein region sequence were not required for the proper synthesis and processing of the viral glycoproteins in vitro. In light of these results, possible expression strategies used by this Phlebovirus M segment RNA are discussed.

Bunyaviridae↗
PubMed1988

Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus.

The genome of bovine viral diarrhea virus (BVDV) contains a single large open reading frame capable of encoding 449 kDa of protein. Short segments from along the length of the molecularly cloned BVDV genome were engineered so as to be expressed as bacterial fusion polypeptides in Escherichia coli. These BVDV analog fusion proteins were used as immunogens to generate a panel of sequence-specific antisera. These antiserum reagents were in turn employed in immunoprecipitation analyses to identify the authentic BVDV protein to which they were directed. The results allowed for the identification and positioning along the genome of BVDV gene products accounting for approximately 83% of the coding capacity of the virus. A preliminary map of the genetic organization of BVDV is presented and discussed.

Antibodies, Viral↗
PubMed1988

Comparisons of the pestivirus bovine viral diarrhoea virus with members of the flaviviridae.

The molecular features of bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus currently classified in the Togaviridae, were examined for characteristics resembling those of the Flaviviridae family. Like flaviviruses, BVDV possesses a single-stranded RNA genome (approx. 4.3 x 10(6) Mr) deficient in a 3' poly(A) tract. This RNA has a single open reading frame spanning the length of the genome in the viral RNA sense (positive polarity), implying an expression strategy involving the processing of a precursor polyprotein. With the exception of several short but significant stretches of identical amino acids within two non-structural proteins, no extended regions of nucleotide or amino acid sequence homology between BVDV and representatives of three serological subgroups of mosquito-borne flaviviruses were noted. However, comparison of the organization of protein-coding domains along the genomes and the hydropathic profiles of amino acid sequences revealed pronounced similarities. It is proposed that Pestivirus, of which BVDV is the prototype member, should no longer be grouped in the Togaviridae family, but rather be considered a genus of non-arthropod-borne viruses within the Flaviviridae.

Amino Acid Sequence↗
PubMed1988

Rift Valley fever virus M segment: use of recombinant vaccinia viruses to study Phlebovirus gene expression.

Recombinant vaccinia viruses were constructed and used in conjunction with site-specific antisera to study the coding capacity and detailed expression strategy of the M segment of the Phlebovirus Rift Valley fever virus (RVFV). The M segment could be completely and faithfully expressed in recombinant RVFV-vaccinia virus-infected cells, the gene products apparently being correctly processed and modified in the absence of the RVFV L and S genomic segments. The proteins encoded by the RVFV M segment included, in addition to the viral glycoproteins G2 and G1, two previously uncharacterized polypeptides of 78 and 14 kilodaltons (kDa). By manipulation of RVFV sequences present in the recombinant vaccinia viruses and use of specific antibody reagents, it was found that the 78-kDa protein initiated at the first initiation codon of the open reading frame and encompassed the entire preglycoprotein and glycoprotein G2 coding sequences. The 14-kDa protein appeared to begin from the second in-phase ATG and was composed of only the preglycoprotein sequences. Both viral glycoproteins G2 and G1 could be synthesized and correctly processed in the absence of the 78- and 14-kDa proteins, as well as a large portion of the preglycoprotein sequences. However, the hydrophobic amino acid sequence immediately preceding the mature glycoprotein coding sequences was required for authentic glycoprotein production. The M-segment expression strategy involving aspects of translational initiation and protein processing are discussed. The functional roles of the 78- and 14-kDa proteins remain unclear.

Bunyaviridae↗
PubMed1987

Forms of pp60v-src isolated from Rous sarcoma virus-transformed cells.

It has previously been shown that an electrophoretic variant form of the Rous sarcoma virus transforming protein, pp60v-src, exists in src-transformed cells. This variant, which was readily observed in vanadate-treated cells, was characterized as possessing extensive amino-terminal domain phosphotyrosine modification. Its appearance was further correlated with increased src-specific protein kinase activity. In this study, we used a src-specific monoclonal antibody (MAb) to resolve immunologic forms of pp60v-src. The MAb was able to distinguish between two populations of typical lower-band pp60v-src and was unreactive with the electrophoretic variant upper-band pp60v-src species. Using serial immunoprecipitations, we resolved four populations of pp60v-src: src protein either immunoreactive or unreactive with the MAb from both untreated and vanadate-treated transformed cells. The pp60v-src in each fraction displayed a distinct phosphoamino acid composition and tryptic phosphopeptide profile. However, analysis of their tyrosyl kinase specific activities showed that the immunologically resolved populations of pp60v-src from a given culture did not differ. Both pp60v-src fractions from vanadate-treated cells exhibited similar kinase specific activities, which were greatly enhanced over those of enzyme preparations from untreated cells. Since the MAb-reactive pp60v-src fraction from vanadate-treated cells lacked the electrophoretic variant upper-band pp60v-src species yet still possessed enhanced enzymatic specific activity, the initially stated correlation between the appearance of the electrophoretic variant src form and increased src kinase activity breaks down. These results suggest that yet to be defined modifications of the src protein may be involved in its functional regulation.

Animals↗
PubMed1986

Messenger RNA of the M segment RNA of Rift Valley fever virus.

The putative messenger RNA (mRNA) of the M segment RNA of the phlebovirus Rift Valley fever virus (RVFV) has been characterized using S1 nuclease mapping and oligonucleotide primer extension procedures. These experiments revealed that the 3' end of the mRNA lacks approximately 112 nucleotides of the M genomic RNA sequences, and that the 5' end of the mRNA possesses all of the sequences present at the 3' end of the M RNA but is further extended beyond the end of the genome by some 12-14 nucleotides of unknown origin. The implications of these data are discussed in relation to the replication and expression strategy of this virus.

Base Sequence↗
PubMed1986

Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus.

Four distinct antigenic determinants along the G2 glycoprotein encoded by the M segment RNA of the Phlebovirus Rift Valley fever virus were localized. These epitopes were defined by four monoclonal antibodies, three of which were capable of neutralizing virus infectivity; one was nonneutralizing. Immunoprecipitation by these monoclonal antibodies of either denatured or native antigen characterized the epitopes as having linear or higher order structure. Molecular cloning of G2 glycoprotein-coding sequences into a bacterial expression plasmid utilizing a beta-galactosidase fusion protein system was employed for epitope localization. A nuclease BAL 31 plasmid expression library, in which processive regions of the 3' end of the G2 glycoprotein coding sequences were deleted, allowed for approximation of the carboxy-terminal limit of the antigenic determinants. Further subcloning of limited G2 polypeptide sequences into the bacterial expression vector permitted more refined localization of the epitopes. The characteristics of the immunoreactivity of these small peptide regions (between 11 and 34 amino acids) produced in bacteria as G2-beta-galactosidase fusion proteins were similar to those of the authentic Rift Valley fever virus G2 glycoprotein. These defined antigenic determinants and their importance in virus infectivity are discussed.

Amino Acid Sequence↗
PubMed1985

Complete nucleotide sequence of the M RNA segment of Rift Valley fever virus.

The entire M RNA segment of the phlebovirus Rift Valley fever virus (RVFV) has been molecularly cloned and the complete nucleotide sequence determined. The RNA is 3884 nucleotides in length, corresponding to a molecular weight of 1.38 X 10(6), having a base composition of 27.3% A, 25.4% G, 27.2% U, and 20.1% C. Sequences present at the 3' and 5' termini of the molecule are largely complementary for some 51 residues and can form a stable duplex structure when the potential secondary structure of the entire molecule is considered. A single major open reading frame, capable of encoding 1206 amino acids (131,845 Da), was found in the viral-complementary sequence ("positive" polarity). Amino-terminal amino acid sequencing of the purified viral glycoproteins G1 and G2 allowed for the positioning of the coding sequences for these polypeptides within this major open reading frame in the following orientation with respect to the genomic M RNA: 3'-G2-G1-5'. From the predicted amino acid composition of the two mature viral glycoproteins, both were found to have a high cysteine content (G2, 6%; G1, 5%). Sequences within the open reading frame capable of encoding up to 23,000 Da of polypeptide were found in addition to those required for the viral glycoproteins. The potential contribution of these sequences to the coding capacity of the M RNA, viral protein processing, and intracellular protein distribution is discussed.

Amino Acid Sequence↗