Biomedical subjects

S K Belzer

Publications and source records attributed to S K Belzer.

6 recordsLinked 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: 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↗

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↗

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↗

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

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↗