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M S Collett

Publications and source records attributed to M S Collett.

At least 55 records · Page 3Linked to original sources
PubMed1985

KBSH parvovirus: comparison with porcine parvovirus.

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.

Animals↗
PubMed1985

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.

Animals↗
PubMed1984

The nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region.

The nucleotide sequence of 7200 bases of encephalomyocarditis (EMC) viral RNA, including the complete polyprotein-coding region, was determined. The polyprotein is encoded within a unique translational reading frame, 6870 bases in length. Protein synthesis begins with the sequence Met-Ala-Thr, and ends with the sequence Leu-Phe-Trp, 126 bases from the 3' end of the RNA. Viral capsid and noncapsid proteins were aligned with the deduced amino acid sequence of the polyprotein. The proteolytic processing map follows the standard 4-3-4 picornaviral pattern except for a short leader peptide (8 kd), which precedes the capsid proteins. Identification of the proteolytic cleavage sites showed that EMC viral protease, p22, has cleavage specificity for gln-gly or gln-ser sequences with adjacent proline residues. The cleavage specificity of the host-coded protease(s) includes both tyr-pro and gln-gly sequences.

Amino Acid Sequence↗
PubMed1984

Enzymatic characteristics of pp60v-src isolated from vanadium-treated transformed cells.

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60v-src. pp60v-src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV-transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60v-src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium-treated cells exhibited similar optimal activity profiles for MgCl2, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis-Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60v-src activity by a vanadium-sensitive protein phosphatase activity.

Animals↗
PubMed1984

Porcine parvovirus DNA: characterization of the genomic and replicative form DNA of two virus isolates.

The genomic and replicative form (RF) DNA of porcine parvovirus (PPV) have been characterized. PPV isolate NADL-8 was found to have a 5000-base single-stranded genome, and a unique strand was encapsidated in virus particles. The RF DNA of isolate NADL-8 was found to be an infectious 5000-base pair (bp) molecule. Select restriction endonuclease sites were mapped along the RF DNA of PPV (NADL-8), and oriented with respect to the viral genomic DNA. The RF DNA of a second isolate of PPV, the less pathogenic, cell culture-adapted NADL-2 virus, was also analyzed. DNA preparations isolated from NADL-2 virus-infected cells contained two viral RF DNA species: a 5000-bp infectious molecule very similar if not identical to the NADL-8 virus RF DNA, and a noninfectious 4700-bp DNA molecule. The 4700-bp RF DNA molecule found in NADL-2 DNA preparations was similar in all respects to the 5000-bp RF DNA, except for a 300-bp deletion in the region encoding the viral capsid proteins. The presence of this defective variant in PPV (NADL-2) virus preparations is discussed in relation to the reduced pathogenic potential of the NADL-2 virus as compared to the NADL-8 virus.

Animals↗
PubMed1984

Characterization of bovine viral diarrhea virus proteins.

Virus-specific proteins were examined in cultured cells infected with bovine viral diarrhea virus. By using antisera obtained from virus-infected animals, three major virus-specific polypeptides with molecular weights of 115,000 (115K), 80K, and 55K were observed. Minor proteins of 45,000 and 38,000 daltons were also noted. Tryptic peptide mapping indicated that the 115K and the 80K polypeptides were structurally related. The 55K protein was glycosylated and appeared not to be related to the 115K and 80K proteins. Pulse-chase experiments failed to demonstrate any procursor-product relationship among any of these proteins, and all three polypeptides were found in purified virion preparations. The significance of these findings with respect to the replication of bovine viral diarrhea virus is discussed.

Animals↗
PubMed1984

Cell-free translation of bovine viral diarrhea virus RNA.

Bovine viral diarrhea virus RNA was translated in a reticulocyte cell-free protein synthesizing system. The purified, 8.2-kilobase, virus-specific RNA species was unable to serve an an efficient message unless it was denatured immediately before translation. In this case, several polypeptides, ranging in molecular weight from 50,000 to 150,000 and most of which were immunoprecipitated by bovine viral diarrhea virus-specific antiserum, were synthesized in vitro. When polyribosomes were used to program cell-free synthesis, mature viral 80,000- and 115,000-molecular-weight proteins were detected; no precursor to the viral 55,000-molecular-weight glycoprotein was noted. The implications of these results with respect to virus-specific protein synthesis are discussed.

Animals↗
PubMed1984

Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.

Animals↗
PubMed1983

Physical modification of purified Rous sarcoma virus pp60v-src protein after incubation with ATP/Mg2+.

Using partially purified enzyme preparations, we show that incubation of the Rous sarcoma virus transforming protein kinase, pp60v-src, with Mg2+ and ATP at concentrations near or above the enzyme's Km for ATP resulted in a physical modification of the pp60v-src polypeptide. Under such conditions, a portion of pp60v-src was converted to a form that migrated more slowly in SDS-polyacrylamide gels than enzyme incubated without ATP or with low concentrations of ATP. Comparative tryptic peptide mapping of pp60v-src incubated with low and high levels of ATP revealed that more extensive tyrosine phosphorylation of the pp60v-src polypeptide occurred at the higher concentrations of ATP. This more extensive phosphorylation was characterized by the appearance of several new phosphorylated tyrosine residues on both the amino-terminal and carboxy-terminal portions of the pp60v-src molecule. The possible consequences of these modifications on the protein kinase activity of pp60v-src, and the functional regulation of retrovirus transforming proteins in general, are discussed.

Adenosine Triphosphate↗
PubMed1983

Porcine parvovirus: virus purification and structural and antigenic properties of virion polypeptides.

Porcine parvovirus (PPV) was extensively purified from infected swine fetal homogenates by CaCl2 precipitation followed by CsCl density centrifugation. Two species of particles possessing PPV-specific hemagglutinating activity were observed banding at densities of 1.39 and 1.30 g/ml, representing full and empty 20-nm virion particles, respectively. Both classes of particles contained three major polypeptides. A, B, and C, with respective molecular weights of 83,000, 64,000, and 60,000. The amount of polypeptide A was similar in both species (approximately 10%); however, the B protein was most abundant in the 1.30-g/ml particles, whereas the C protein was the major polypeptide found in the 1.39-g/ml particles. Antisera generated to each sodium dodecyl sulfate-polyacrylamide gel-purified virion structural protein had reactivities qualitatively similar to those of conventional antisera raised against intact PPV in a variety of standard serological assays. The antisera generated against the individual sodium dodecyl sulfate-denatured PPV polypeptides were able to react with native, intact PPV virions and were capable of neutralizing virus infectivity.

Animals↗
PubMed1983

Specific proteolytic fragmentation of p60v-src in transformed cell lysates.

Work involving the transforming protein, p60v-src, of Rous sarcoma virus has resulted in the extensive characterization of its protein structure and associated phosphotransferase activity. However, in many investigations proteolytic fragments (principally p52v-src) of the src protein are actually studied. Here, we emphasize potential problems in the interpretation of experimental results in which the proteolytic fragmentation of p60v-src may be involved and offer several means for the complete prevention of this p60v-src degradation.

Animals↗
PubMed1983

Characterization of virus-specific RNA synthesized in bovine cells infected with bovine viral diarrhea virus.

Infection of bovine kidney cells with bovine viral diarrhea virus resulted in the synthesis of a single species of virus-specific RNA. Electrophoresis of this RNA on agarose-urea and agarose-formaldehyde gels indicated that it had a molecular weight of 2.9 X 10(6), corresponding to 8,200 bases (8.2 kilobases). This 8.2-kilobase RNA was resistant to RNase A treatment at 1 microgram/ml but was digested at higher concentrations of RNase (10 micrograms/ml). Sedimentation on neutral sucrose gradients indicated that the majority of this RNA (98%) sedimented at 21S, with a small amount sedimenting at 33S. Sedimentation on formaldehyde-containing sucrose gradients resulted in the conversion of all of the RNA to the faster-sedimenting form. At no time after infection were we able to detect virus-specific RNA species of lower molecular weight than the 8.2-kilobase RNA. The implications of these findings with respect to the means of replication of various togaviruses are discussed.

Animals↗
PubMed1983

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

pp60v-src, the product of the Rous sarcoma virus src gene, was partially purified by immunoaffinity chromatography from extracts of Rous sarcoma virus-transformed field vole cells. Incubation of this preparation with ATP plus Mg2+ and subsequent repurification by chromatography on hexylamine-agarose resulted in a net increase in the specific activity of the src protein kinase. This increase in phosphotransferase activity was detected by using a variety of substrates including casein, tubulin, and a 34,000-dalton protein presumed to be an in vivo target substrate of pp60v-src. In all cases, the phosphorylation was at tyrosine residues, and the kinase activity was inhibited by preincubation of the enzyme with immunoglobulin G prepared from tumor-bearing rabbit sera. The implications of these results for the regulation and control of pp60v-src-associated kinase activity are discussed.

Adenosine Triphosphate↗
PubMed1980

Towards a molecular description of cell transformation by avian sarcoma virus.

The avian sarcoma virus transforming gene product has been identified and partially purified from extracts of transformed cells. It is a phosphoprotein with a relative molecular mass of 60 000 (pp60src) with two major sites of phosphorylation. pp60src appears to be a cyclic-AMP-independent protein kinase as judged by protein phosphorylation with partly purified fractions. The specificity of the phosphorylation observed was judged by inhibition with anti-pp60src IgG but not by normal IgG and by the fact that the protein kinase activity isolated from ts transformation-mutant infected cells was more thermolabile than that from wild-type transformed cells, thus showing more directly the origin of the enzymic activity. A cellular protein substrate of pp60src has been identified as a 34 000 molecular mass protein. These data together suggest that protein phosphorylation by pp60src may be a function of the molecule that plays a major role in transformation.

Alpharetrovirus↗
PubMed1980

Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine.

The protein responsible for malignant transformation by avian sarcoma viruses (ASVs) has been identified as a phosphoprotein of molecular weight 60,000 designated pp60src (refs 1--4). It has been suggested that this protein has a functional role in cellular transformation involving the phosphorylation of cellular proteins, for it was discovered that specific immunoprecipitates from ASV-transformed cells that contain pp60src catalysed the transfer of phosphate from [gamma-32P]ATP to the heavy chain of rabbit immunoglobulin. Additional studies involving the cell-free synthesis of the ASV src protein further demonstrated that the presence of the src polypeptide correlated with that presence of a phosphotransferase activity. Our studies, involving the biochemical purification of this protein, have demonstrated that the ASV-transforming gene product, pp60src, is itself a protein kinase. We have purified the pp60src protein approximately 5,000-fold using either conventional ion-exchange chromatography or immunoaffinity chromatography. The resultant partially purified preparations contain a cyclic AMP-independent protein kinase activity. We report here that the soluble phosphotransferase activity of partially purified pp60src results in the phosphorylation of exclusively tyrosine residues in a variety of proteins that serve as substrates.

Actins↗
PubMed1980

Enzymatic synthesis of deoxyribonucleic acid by the avian retrovirus reverse transcriptase in vitro: optimum conditions required for transcription of large ribonucleic acid templates.

In this communication, we present data which describe optimum conditions for reverse transcription of large ribonucleic acid (RNA) templates into deoxyribonucleic acid (DNA) transcripts by the avian retrovirus reverse transcriptase in vitro. In contrast to previous studies, we have optimized all of the reaction components with respect to their influence on the size of DNA transcripts rather than the incorporation of radio-labeled deoxynucleoside triphosphates into acid-insoluble DNA product. The most dramatic effect on uninterrupted reverse transcription is the presence of physiological concentrations (i.e., 148 mM) of monovalent cation in the reaction mixture, although all of the components of the reaction influence the size of the DNA transcripts synthesized to some extent. The enzymatic conditions described herein for the uninterrupted reverse transcription of large RNA templates (greater than 1000--2000 nucleotides) are superior to those described previously because they are reproducible, do not require the presence of ribonuclease inhibitors, and do not result in the precipitation of components of the reaction mixture during incubation.

Avian Leukosis Virus↗
PubMed1980

Molecular events in cells transformed by Rous Sarcoma virus.

The Rous sarcoma virus (RSV) transforming gene product has been identified and characterized as a phosphoprotein with a molecular weight of 60,000, denoted pp60src. Partially purified pp60src displays a closely associated phosphotransferase activity with the unusual specificity of phosphorylating tyrosine residues in a variety of proteins. That the enzymatic activity observed is actually encoded by the RSV-transforming gene is indicated by the comparison of the pp60src-protein kinase isolated from cells tranformed by a wild-type RSV or by a RSV temperature-sensitive transformation mutant; these experiments revealed that the latter enzyme had a half-life of 3 min at 41 degrees C, whereas that of the wild-type enzyme was 20 min. Evidence is now beginning to accumulate showing that viral pp60src expresses its protein kinase activity in transformed cells as well as in vitro because at least one cellular protein has been identified as a substrate for this activity of pp60src. Although the protein kinase activity associated with pp60src is itself cyclic AMP (cAMP) independent, the molecule contains at least one serine residue that is directly phosphorylated by the cellular cAMP-dependent protein kinase, thus suggesting that the viral transforming gene product may be regulated indirectly by the level of cAMP. The significance of this latter observation must be regarded from the point of view that the RSV src gene is apparently derived from a normal cellular gene that seemingly expresses in normal uninfected cells a phosphoprotein structurally and functionally closely related to pp60src. This celluar protein, found in all vertebrate species tested, also is a substrate for a cAMP-dependent protein kinase of normal cells, and, therefore, may be evolved to function in a regulatory circuit involving cAMP.

Animals↗