Biomedical subjects
J R Stephenson
Publications and source records attributed to J R Stephenson.
Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.
Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus.
Abelson murine leukemia virus P120: identification and characterization of tyrosine phosphorylation sites.
Tryptic peptides containing two major in vivo P120gag-abl tyrosine phosphorylation acceptor sites were identified, phosphorylated in vitro, and purified to homogeneity. The tyrosine site in peptide a is localized at a position six residues distal to its trypsin cleavage site, whereas the tyrosine acceptor site in peptide b is at residue seven. A third peptide, c, contains an amino-terminal phosphotyrosine residue: phosphorylation of this latter peptide only occurs to a significant extent in vivo.
Civilian-military contingency hospital system: a cooperative effort to provide medical care in the case of sudden military conflict.
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Human transforming growth factors induce tyrosine phosphorylation of EGF receptors.
Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.
Amino- and carboxyl-terminal sequences of proteins coded by gag gene of endogenous baboon and cat type C viruses.
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Genetic variation of neurotropic and non-neurotropic murine coronaviruses.
The murine coronavirus strains MHV JMH, MHV 1, MHV 2, MHV 3 and MHV A 59 were tested for their neurovirulence in weanling rats. The strain JHM was found to be highly neurovirulent for weanling rats, whereas the other strains were not, or only slightly, neurovirulent. MHV 1 caused no lesions in weanling rats. The other strains (MHV 2, MHV 3 and MVH A59) induced predominantly subclinical infections in weanling rats as demonstrated by an increase of antibodies and inflammatory lesions in the liver. Analysis of these strains by cross-neutralization revealed variable degrees of antigenic relationship between these viruses which were not related to their neurovirulence. However, when these strains were compared by analysing the T1-RNase-resistant oligonucleotides of virion RNA, the highly neurovirulent strain JHM was found to differ significantly in its nucleotide sequence from the other less-neurovirulent strains.
Persistent and lytic infections with SSPE virus: a comparison of the synthesis of virus-specific polypeptides.
The synthesis of virus-specific polypeptides and messenger RNA in cell cultures persistently infected with an isolate of measles virus from a patient with subacute sclerosing panencephalitis (SSPE) has been compared to that found in a lytic infection with the homologous virus. The persistent infection described here was chosen as its biological characteristics reflect those of virus-infected brain cells from SSPE patients. The synthesis of H, N and possibly F protein was seen in both lytic and persistent infections, but the synthesis of M protein was only detected in the lytic infection. However, messenger RNA isolated from either the lytic or persistent infection directed the synthesis in a cell-free translation system of all structural polypeptides, including M, and also three non-structural polypeptides, with mol. wt. of 34 000, 30 000 and 18 000. Messenger RNAs coding for the virus-specific polypeptides were also shown to be polyadenylated. In addition, those polypeptides made in vitro which were antigenically related to the haemagglutinin, demonstrated structural changes after passage through a persistent infection.
Antigenic characterization of measles and SSPE virus haemagglutinin by monoclonal antibodies.
Hybrid cells secreting monoclonal antibodies directed against the haemagglutinin (H) protein of measles virus (Edmonston) were produced by fusion of mouse myeloma cells with spleen cells derived from immunized mice. Measles antibodies secreted by these cells were tested for their ability to react with measles virus in immunoprecipitation experiments and assays of binding, neutralization, haemagglutination inhibition and haemolysin inhibition. On this basis 21 out of 75 hybridomas could be defined and divided into five functional groups with different properties. However, when tested against other measles virus strains, including those isolated from subacute sclerosing panencephalitis (SSPE) patients, normalized radioimmunoassay (RIA) binding titres showed that the extent to which a given antibody bound could vary greatly with the virus strain examined. Moreover, the biological actions within a group were found to be very heterogeneous, even when high antibody binding titres were observed. These results suggest that different measles virus strains, which are not distinguishable by polyvalent sera, do in fact possess antigenic differences. Furthermore, the functional significance of a given virus epitope may vary from strain to strain. Hybridoma antibodies were also used to demonstrate the occurrence of antigenic changes within the H polypeptide of SSPE virus during the course of non-productive, persistent infection in vitro.
Involvement of a high-molecular-weight polyprotein translational product of Snyder-Theilen Feline sarcoma virus in malignant transformation.
The previously described high-molecular-weight polyprotein major translational product of the Snyder-Theilen strain of feline sarcoma virus (FeSV) was shown to possess protein kinase activity with specificity for tyrosine acceptor sites. Cells transformed by Snyder-Theilen FeSV exhibited constitutively elevated levels of phosphotyrosine and a concomitant reduction in epidermal growth factor (EGF) binding sites. By endpoint cloning in microtiter plates, a number of transformation-defective (tf) mutants of the Snyder-Theilen strain of FeSV were isolated. Mink cells nonproductively infected by such mutants were morphologically nontransformed, failed to grow in soft agar, bound EGF as efficiently as control mink cells, and lacked rescuable transforming virus. Although the level of expression of the major viral polyprotein translational product in td mutant-infected clones was comparable to that of wild-type (wt) transformants, the polyprotein in mutant clones lacked detectable protein kinase activity and total cellular phosphotyrosine levels were not elevated significantly above control values. Of a large number of wt Snyder-Theilen FeSV-transformed mink cell clones isolated, the majority were found to revert to a nontransformed morphology upon continuous passage in cell culture. Such nontransformed variants, as well as a Gardner FeSV-transformed mink cell revertant, lacked detectable polyprotein expression and exhibited levels of phosphotyrosine and EGF binding similar to those of control mink cells. These findings provide strong evidence favoring the involvement of the Snyder-Theilen FeSV-encoded high-molecular-weight polyprotein and its associated tyrosine-specific protein kinase activity in transformation.
Differences in mechanisms of transformation by independent feline sarcoma virus isolates.
The Gardner and Snyder-Theilen isolates of feline sarcoma virus (FeSV) have previously been shown to encode high-molecular-weight polyproteins with a transforming function and an associated tyrosine-specific protein kinase activity. Cells transformed by these viruses exhibited morphological alterations, elevated levels of phosphotyrosine, and a reduced capacity for binding epidermal growth factor. In addition, polyproteins encoded by both of these FeSV isolates bound to, and phosphorylated tyrosine acceptor sites within, a 150,000-molecular-weight cellular substrate (P150). McDonough FeSV-transformed cells resembled Gardner and Snyder-Theilen FeSV transformants with respect to morphological changes and a reduced capacity for epidermal growth factor binding. in contrast to the other two FeSV isolates, however, McDonough FeSV encoded as its major translational product a high-molecular-weight polyprotein with probable transforming function but without protein kinase activity detectable under similar assay conditions. Moreover, total cellular levels of phosphotyrosine remained unaltered in McDonough FeSV-transformed cells, and the major McDonough FeSV polyprotein translational product lacked binding affinity for P150. These findings argue for differences in the mechanisms of transformation by these independently derived FeSV isolates.
Snyder-Theilen feline sarcoma virus P85 contains a single phosphotyrosine acceptor site recognized by its associated protein kinase.
Cells nonproductively transformed by a variant of the Snyder-Theilen strain of feline sarcoma virus (FeSV) expressed an 85,000-dalton polyprotein (P85) with associated tyrosine-specific protein kinase activity. We identified within this polyprotein a single tyrosine acceptor site for its enzyme activity. This acceptor site, as well as two serine phosphorylation sites localized with the p12 structural component of Snyder-Theilen FeSv P85, was phosphorylated in cells nonproductively transformed by Snyder-Theilen FeSv. In contrast, infection by Snyder-Theilen FeSV transformation-defective mutants resulted in phosphorylation only of the two serine acceptor sites, indicating phosphorylation of the tyrosine acceptor site to be transformation specific. In addition, we describe in vitro labeling conditions, using unfractionated cell extracts, which resulted in preferential phosphorylation of the single Snyder-Theilen FeSV tyrosine-specific acceptor site.
Identification of tryptic peptides unique to a 110,000-molecular weight polyprotein encoded by the T-8 isolate of murine leukemia virus.
Mink cells nonproductively-infected with the weakly-transforming T-8 isolate of murine leukemia virus (MuLV) express a 110,000 mol wt polyprotein designated T-8 P110. By immunoprecipitation analysis, T-8 P110 is shown to contain AKR-MuLV amino terminal gag gene-specific components (p15, p12) but to lack p30, p10, gp70, and p15(E) antigenic determinants. These observations are further substantiated by tryptic peptide analysis indicating T-8 P110 to share approximately six lysine-containing tryptic peptides with AKR-MuLV Pr65gag, and none with AKr-MuLV Pr82env. Furthermore, of seven methionine-containing T-8 P110 tryptic peptides, at least four can be conclusively shown not to be present in either AKr-MuLV Pr180gag/pol or Pr82env. A clonal mink cell line nonproductively infected by T-8, and expressing high levels of P110, although not morphologically transformed, is shown to lack elevated levels of tyrosine-specific protein kinase activity and reduction of epidermal growth factor binding sites characteristic of cells transformed by many other RNA-transforming viruses. These findings argue either that the T-8 viral genome contains acquired cellular sequences encoding a portion of P110, or that T-8 P110 represents an inphase deletion of AKR-MuLV Pr180gag/pol with extensive posttranlational modification and that an as yet unidentified protein is responsible for T-8 associated transformation.
Feline sarcoma virus P115-associated protein kinase phosphorylates tyrosine. Identification of a cellular substrate conserved during evolution.
The Gardner strain of feline sarcoma virus (FeSV) has previously been shown to encode as its major translational product, a phosphoprotein of molecular weight (Mr) = 115,000 (P115) with an associated protein kinase activity and probable transforming function. In the present study, we demonstrate that this enzyme specifically phosphorylates tyrosine and that Gardner FeSV-transformed mink and rat cells exhibit constitutively higher levels of phosphotyrosine than nontransformed control cells. In addition, we identify a Mr = 150,000 cellular substrate for the FeSV P115-associated protein kinase activity. This latter protein, designated P150, is expressed in cells of a number of mammalian species including cat, mink, dog, rat, mouse and human, possesses broadly reactive interspecies antigenic determinants, and exhibits binding affinity for FeSV P115. Although P150 exhibits an associated protein kinase activity, this activity differs from the FeSV P115-associated enzyme in that it phosphorylates serine and threonine rather than tyrosine. On the basis of immunologic criteria and tryptic peptide analysis, the possibility that P150 is structurally related to Gardner FeSV P115 is excluded. As a consequence of its affinity for FeSV P115, P150 is efficiently incorporated into FeSV pseudotype virions, providing a source for its purification and preparation of antisera for study of its expression in mammalian cells.
Search for canine-distemper-virus antibodies in multiple sclerosis. A detailed virological evaluation.
Sera and cerebrospinal fluid samples from multiple-sclerosis (MS) patients, matched controls, and subacute sclerosing panencephalitis (SSPE) patients were analysed for the presence of antibodies specific for measles and canine distemper virus (CDV). Virus-specific antibodies were identified by haemagglutination inhibition, neutralisation, and immune precipitation in combination with polyacrylamide-gel electrophoresis. SSPE and MS patients had a higher antibody activity towards measles virus than controls. Moreover, human sera partly neutralised CDV; this was attributed to antibody activity against CDV proteins, antigenically related to those of measles virus. However, CDV-specific antibodies were not found in the samples tested. These findings indicate that MS can develop without a preceding CDV infection.
Abelson murine leukaemia virus transformation involves loss of epidermal growth factor-binding sites.
Malignant transformation by mammalian RNA sarcoma viruses has previously been shown to involve a reduction in receptor sites for a well characterized 6,000-molecular weight (MW) growth-promoting substance, designated epidermal growth factor (EGF). Although Abelson murine leukaemia virus (AbLV) resembles sarcoma viruses in its ability to transform embryo fibroblasts in cell culture, AbLV induces a rapid B-cell lymphoid leukaemia rather than fibrosarcomas in vivo. The major translational product of AbLV is a highly phosphorylated polyprotein of MW 120,000 which exhibits an associated tyrosine-specific protein kinase activity and probable transforming function. We show here that AbLV transformation resembles transformation by RNA sarcoma viruses with respect to the abolition of EGF-binding sites. EGF binding is restored to control levels following loss of polyprotein expression in morphological revertants of AbLV-transformed clones and remains uninfluenced in cell lines infected with transformation-defective (td) AbLV mutants encoding polyproteins deficient in protein kinase activity. These findings indicate that AbLV transformation involves a polyprotein-associated, tyrosine-specific protein kinase activity which mediates its effect through a mechanism resulting directly or indirectly in the abolition of EGF-binding sites.
Feline sarcoma virus polyprotein P115 binds a host phosphoprotein in transformed cells.
Several independent isoltes of feline sarcoma virus (FeSV) have been described. Such viruses are apparently derived by genetic recombination between feline leukaemia virus (FeLV) genomic RNA and host cellular genetic sequences with transforming potential. Two FeSV isolates, one originally described by Gardner and the second by Snyder-Theilen, have been shown to encode polyproteins of around 115,000 molecular weight. Both polyproteins contain FeLV structural components (p15, p12) at their amino terminus in addition to nonstructural carboxyl terminal components encoded by acquired sequences within the FeSV genome. We have previously shown that Gardner FeSV P115 contains multiple sites of phosphorylation within its nonstructural component and possesses an associated protein kinase activity. In the present study we describe the expression in cells derived from a number of mammalian species, of a highly conserved celklular phosphoprotein with binding affinity for Gardner FeSV P115. This protein, designated P150, exhibits an associated protein kinase activity and is immunologically and structurally distinct from polyproteins encoded by the Gardner or Snyder-Theilen strains of FeSV.
Anemia- and polycythemia-inducing isolates of Friend spleen focus-forming virus. Biological and molecular evidence for two distinct viral genomes.
Two distinct clones of Friend spleen focus-forming virus (SFFV), differing in their erythroleukemic potential, are described. These isolates have been cloned free of their associated helper viruses and shown to be replication-defective. Both SFFV isolates have been rescued from rat fibroblast nonproducer cell clones with cloned replication-competent viruses, F-MuLVA and F-MuLVP, obtained from the anemia- or polycythemia-inducing isolates of Friend virus complex, respectively. These rescued viruses induce a rapid proliferative disease associated with the appearance of macroscopic spleen foci and splenomegaly. In addition, each is subject to regulation by the W, Steel (Sl), and Fv-2 host gene loci. These two isolates of SFFV can, however, be distinguished by both biological and molecular criteria. Friend SFFVP induces a rapid polycythemia associated with the appearance of large numbers of erythropoietin (EPO)-independent erythroid colony-forming cells in the marrow and spleen. In contrast, SFFVA induces a rapid anemia associated with a progressive decrease in the number of EPO-dependent erythroid colony-forming cells in marrow, and a rapid increase in the number of EPO-dependent erythroid colony-forming cells in spleen. Furthermore, the nature of the disease induced by the two isolates of SFFV is independent of the Friend helper virus: SFFVP, rescued from a nonproducer cell clone with either F-MuLVA or F0MuLVP, induced a polycythemic transformation, whereas SFFVA, rescued with either F-MuLVA or F-MuLVP, induced an anemic transformation. The two Friend SFFV isolates can also be discriminated on the basis of translational products encoded by their gag and env genes: SFFVP encodes the amino-terminal gag-gene protein p15, whereas SFFVA encodes the gag-gene proteins p15, p12, and p30. In addition, the SFFV isolates encode nonidentical 55,000-mol wt env gene-related proteins that can be distinguished by analysis of their methionine-containing tryptic peptides.