Heinz Bauer

Early changes in the distribution and organization of microfilament proteins during cell transformation

During the onset of transformation, Rous sarcoma virus-infected cells undergo characteristic morphological changes that reflect the biochemical events induced by the viral src gene. Temperature downshift experiments using chick embryo cells infected with transformation-defective temperature-sensitive viral mutants have shown two major morphological changes occurring at different times in the transformation process: ruffle-like flowers appear on the dorsal cell surface as early as 15 min after temperature shift, while later, between 6 and 12 hr, cytoskeletal stress fibers disappear and the cells round up. We report that flowers contain large accumulations of the cytoskeletal proteins actin, alpha-actinin, myosin and tropomyosin. Furthermore, since flowers stain very intensely with fluorescein-labeled phalloidin, a cyclopeptide that selectively binds to F-actin and not to G-actin, we suggest that these structures result from an early reorganization of microfilaments.

Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src

To study the function of pp60src, the transforming protein encoded for by Rous sarcoma virus, we have raised antibodies against synthetic oligopeptides corresponding to the primary structure of pp60src. All eight investigated peptides were immunogenic in rabbits, and four induced pp60src-specific antibodies. We screened tumor-bearing rabbit (TBR) sera for antibodies against the peptides; this revealed that five out of six of the peptides, chosen according to a high hydrophilicity plot, were related to epitopes of native pp60src, in contrast to two peptides of low hydrophilicity, which contained a cleavage site for protease. Antibodies against three of the peptides appeared to react with the kinase-active site of pp60src, as these antibodies were phosphorylated in their heavy chain upon immune precipitation. Antibodies against two of the peptides, in contrast to the others, did not precipitate pp60src when this molecule was complexed with two cellular proteins, pp50 and pp90. This observation allows speculation about the location of the pp60src site involved in the formation of this complex.

Molecular events during the interaction of envelopes of myxo- and RNA-tumor viruses with cell membranes. A 270 MHz 1H nuclear magnetic resonance study

Abstract The nuclear magnetic resonance (NMR) spectra of chick embryo cells have been analyzed after exposure to Newcastle disease virus (NDV). Virions that contained the envelope glycoproteins in the cleaved form and, thus, had full biological activity have been compared to virions that had reduced infectivity due to the presence of uncleaved glycoprotein F. After exposure to infectious virus, drastic changes occurred in the signals assigned to choline and the hydrocarbon chains of fatty acids. These observations are interpreted to demonstrate alteration of the fluid lipid bilayer structure of the cell membranes. This is compatible with the concept of membrane fusion as a penetration mechanism for NDV. Virus containing uncleaved F glycoprotein did not alter the NMR spectra. This indicates that infection is blocked at the stage of penetration. Similar, though less pronounced, differences have been observed when the effects of highly infectious influenza virus containing the hemagglutinin in the cleaved form were compared to the effects of virus which had a lower infectivity due to the presence of uncleaved hemagglutinin. Thus, it appears that the hemagglutinin of influenza virus is involved in penetration and that cleavage is necessary for this function. Alterations of the NMR spectra of the membrane lipids have also been observed when susceptible chick embryo cells (C/E) were infected with Rous sarcoma virus of subgroup B. Such alterations did not occur when nonsusceptible cells (C/B) were used. Thus, infection appears to be blocked again at the stage of penetration.

A comparative study of the avian reticuloendotheliosis virus: relationship to murine leukemia virus and viruses of the avian sarcoma-leukosis complex.

Abstract Reticuloendotheliosis virus (REV) with several related viruses comprises a group of C-type, avian RNA tumor viruses which is distinct from the avian leukosis-sarcoma virus (ALSV) complex. The emphasis of the present investigation has been on the comparison of the properties of REV with those of a model mammalian RNA tumor virus, murine leukemia virus (MuLV), and with those of the ALSV. The structure and pathway of maturation of these viruses has been examined using electron microscopy. Conclusions derived from this work indicate that while the immature particles of REV can be morphologically distinguished from both MuLV and ALSV, the mature REV particle is very like that of MuLV and quite different from that of ALSV. The properties of the purified DNA-polymerase of these viruses were analyzed with the following significant findings: 1) Unfrozen, purified disrupted REV, but not purified REV-DNA-polymerase is enzymatically active using natural viral RNA as template-primer; 2) the DNA-polymerase copurifies with an RNase-H activity which probably resides on the same polypeptide; 3) the size of the DNA-polymerase-RNase-H complex is indistinguishable from that of the MuLV, a single polypeptide of 84,000 daltons; 4) the divalent cation preference of the REV-DNA polymerase, like that of MuLV, but unlike that of ALSV, is for Mn 2+ ; and 5) serological cross-reaction between the DNA-polymerase of REV and ALSV could not be demonstrated. Apart from these structural and biochemical analogies, no direct relationship between REV and MuLV has been established. Infectivity of REV in two strains of mouse cells could not be demonstrated. Immunodiffusion tests for reaction of purified, disrupted REV with antisera specific for ALSV structural components and for the interspecies specific reaction characteristic of mammalian RNA tumor virus p 30 protein were uniformly negative. After consideration of all the available data, it seems that the REV must be considered a distinct group of avian RNA tumor viruses with significant structural similarities to mammalian viruses, but nonetheless differing antigenic determinants.

Biochemical characterization of tumor-specific cell surface antigens on avian oncornavirus transformed cells

Abstract A sensitive indirect immune precipitation method coupled with polyacrylamide-gel electrophoresis was used to detect new antigens from avian oncornavirus-transformed chicken embryo cells (CEC). A total of four antigens were recognized by this method. Two of these antigens appeared to be identical to the gp85 and gp37 components of the virus envelope. The other two antigens were distinct from the viral envelope antigens on the basis of both size and antigenicity, and were group specific for the avian oncornavirus system. The larger nonvirion antigen was detectable on transformed CEC but absent from normal or productively infected nontransformed CEC and therefore was clearly tumor specific. Because of the weak detectability of the smaller nonvirion antigen, its tumor specificity could not be decided. The tumor-specific antigen was a glycoprotein of 100,000-dalton apparent molecular weight, whereas the smaller nonvirion antigen was 32,000 daltons and probably also a glycoprotein. The 100,000-dalton tumor-specific antigen was found on the cell surface and therefore represented a tumor-specific cell surface antigen (TSSA).

The differential expression of the cellular src-gene product pp60src and its phosphokinase activity in normal chicken cells and tissues

Chick embryos of different ages and adult chickens were examined for the expression of pp60c -src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. In any brain extract, the pp60c -src kinase activity was always high, whereas muscle extracts of embryos show an age-dependent decrease in kinase activity. Adult animals show either no or barely measurable activity in muscle tissue. In contrast, liver cell extracts of embryos show an age-dependent increase in pp60c -src kinase activity, with adult chickens displaying the highest activity, very similar to that found in brain extracts. This demonstrates that increased expression of c-src is not necessarily correlated with cell proliferation, but suggests that, at an early stage of differentiation of mesenchymal cells, the relatively high expression of c-src could be responsible, at least in part, for the control of cell metabolism and proliferation.

Expression of tumor-specific surface antigens on cells infected with temperature-sensitive mutants of avian sarcoma virus

Abstract Tumor-specific surface antigens (TSSA) have been found in all avian sarcoma virus-transformed cells so far examined. We have studied the expression of these antigens in cells infected with mutant viruses and in which a phenotypically normal or transformed state depends on the temperature of incubation. We found that in cells infected with these mutants, all of which are defective in the ability to induce host cell transformation at the nonpermissive temperature, there are differences in the expression of TSSA. The questions whether TSSA are viral-coded functions and whether they are directly involved in the maintenance of the transformed phenotype are discussed in the light of our results.

Biological and biochemical characterization of a new isolate of feline sarcoma virus: Theilen-pedersen (TP1-FeSV)

A new feline sarcoma virus designated Theilen-Pedersen (TP1-FeSV) has been isolated from a spontaneous, slowly growing fibrosarcoma of a domestic short-haired 4-year-old castrated cat. The virus codes for a gag-onc fusion protein of 83,000 molecular weight phosphorylated in vivo at serine, threonine, and tyrosine residues. Cells transformed in vitro with TP1-FeSV exhibit five- to 10-fold elevated levels of phosphotyrosine over FeLV-infected cells. The gag-onc polyprotein has associated with it a tyrosyl protein kinase activity which in vitro results in autophosphorylation of the molecule at tyrosine residues. The fusion protein cannot be labeled metabolically with [3H]glucosamine and tunicamycin has no effect on the electrophoretic mobility of the in vivo [32P]orthophosphate-labeled fusion protein. The fusion protein, in common with the gag precursor Pr65gag, can be metabolically labeled with palmitic acid.

pp60c-src is a substrate for phosphorylation when cells are stimulated to enter cycle

The endogenous cellular oncogene products, pp60 c‐src , exhibits a protein kinase activity, but is itself a phosphoprotein. Based on the assumption that pp60 c‐src might play a role in the control of cell proliferation, we have studied its behaviour as a substrate for phosphorylation known to occur when quiescent, serum‐deprived cells are stimulated to enter cell cycle following addition of either serum, platelet‐derived growth factor or the phorbol ester derivative, 12‐0‐tetradecanoyl‐phorbol‐13‐acetate. For this purpose a partial purification of pp60 c‐src on DEAE ion‐exchange chromatography was combined with immune precipitation. A 2–4‐fold increase in serine phosphorylation of pp60 c‐src was consistently observed after stimulation of quiescent cells to growth.

The expression of pp60src and its associated protein kinase activity in cells infected with different transformation-defective temperature-sensitive mutants of rous sarcoma virus

Abstract A set of five transformation-defective temperature-sensitive mutants of Rous sarcoma virus has been used to investigate the relation between pp60 src its associated protein kinase activity, and expression of the transformed phenotype. In radioimmune competition experiments, the levels of pp60 src induced by the mutants did not vary by more than a factor of two, either among the mutants at a given temperature or between nonpermissive and permissive temperatures for a given mutant. The mutants fell into two distinct classes with respect to the temperature conditional expression of pp60 src -associated kinase activity. Three mutants (GI 201, GI 202, and GI 251) induced two- to fivefold higher levels of pp60 src -associated kinase activity at the permissive temperature. The other mutants GI 203 and 253 induced only very low levels of pp60 src -associated kinase at either temperature. The pp60 src -associated kinase activity induced by GI 201, 202, and 251 at the permissive temperature was significantly more heat labile in vitro than that of the wild type. Furthermore, downshift of the mutant-infected cells to the permissive temperature resulted in a rapid increase (within 15 min) in the pp60 src -associated kinase activity only with mutants GI 201, GI 202, and GI 251, i.e., only with those mutants having an elevated activity at the permissive temperature. The results taken as a whole suggest that there is not a simple relationship between pp60 src , pp60 src -associated kinase activity, and transformation and support the idea of multifunctionality of the src gene product.