Henry R. Bose

Transformation by reticuloendotheliosis virus: Development of a focus assay and isolation of a nontransforming virus

Abstract A focus assay for quantitating in vitro transformation by oncogenic reticuloendotheliosis virus (REV) has been developed in Japanese quail embryo fibroblast cultures. The titer of the transforming virus detected in the in vitro focus-forming assay correlates with the development of reticuloendotheliosis in chickens. The titration pattern of REV focus formation appears to follow two-hit kinetics. A nontransforming member has been identified in the REV stocks and is designated reticuloendotheliosis-associated virus (REV-A). The non-transforming virus, REV-A, is present in REV stocks in 100- to 1000-fold excess over the transforming member. REV-A causes an initial cytopathic effect in chick and quail embryo fibroblast cultures. The surviving cells continue to divide leading to the development of a persistently infected culture. The persistently infected cultures are morphologically indistinguishable from uninfected avian fibroblast cultures. REV-A interferes with superinfection by the oncogenic member. The addition of REV-A to oncogenic preparations of REV increases focus formation and changes the titration pattern from two-hit to one-hit kinetics indicating that REV is defective.

Induction of stress proteins in Sindbis virus- and vesicular stomatitis virus-infected cells

Two proteins which are related to certain proteins induced by hyperthermia (heat shock proteins; hsp) are synthesized during lytic infection of chick embryo (CE) cells by Sindbis virus or vesicular stomatitis virus (VSV). Incubation of the infected cells at elevated temperature further increased the rate of synthesis of these proteins. The stress proteins induced by Sindbis virus had different mobilities on SDS-polyacrylamide gels compared to related stress proteins induced in mock-infected CE cells. Induction of the stress proteins in Sindbis virus- and VSV-infected CE cells was actinomycin D sensitive. Kinetic studies indicated that induction of the stress proteins is an early event during infection. The lytic virus-induced selective termination of host protein synthesis did not affect the synthesis of these proteins. Furthermore, the synthesis of these virus-induced stress proteins was resistant, relative to the synthesis of most host proteins, to alterations in the intracellular concentrations of Na+ and K+. The synthesis of a protein related to a major low-molecular-weight hsp of CE cells was not induced after Sindbis virus or VSV infection. Immunoprecipitation experiments and sedimentation analyses demonstrated that significant levels of the capsid protein (C) of Sindbis virus and nucleocapsid protein (N) of VSV are physically associated with a hsp in lysates of infected CE cells.

Expression of the v-rel oncogene in reticuloendotheliosis virus-transformed fibroblasts.

Reticuloendotheliosis virus (REV-T) induces a rapidly fatal lymphoma in chickens through the expression of its oncogene, v-rel, REV-T also morphologically transforms avian fibroblasts in vitro. These transformed cells displayed limited anchorage-independent growth and reached higher saturation density than uninfected or REV-A-infected fibroblasts. Morphologically transformed fibroblasts were tumorigenic when injected into the wing web of chickens. In transformed fibroblasts, the v-rel oncogene was expressed as a 57 kDa phosphoprotein with a half-life of 2 to 4 hr. A cellular phosphoprotein of about 40 kDa was also observed in immunoprecipitates of transformed fibroblasts. The subcellular location of the v-rel-encoded protein was determined using cell fractionation procedures and immunofluorescent staining. In acutely infected, nontransformed fibroblasts, pp57v-rel was associated with the nuclear region, but in morphologically transformed cells the v-rel protein was found in the cytoplasm. These observations suggest that the expression of the v-rel oncogene is insufficient for transformation and that the cellular localization of this transforming protein to the cytoplasm may be required for the progression to an altered cell phenotype in avian fibroblasts.

Helper viruses associated with avian acute leukemia viruses inhibit the cellular immune response

Abstract The cellular immune competence of lymphocytes obtained from birds infected with acute or chronic leukemia viruses was studied. The in vitro blastogenic response of thymus-derived lymphocytes (T cells) to the mitogens phytohemagglutinin (PHA) and connanavalin A (Con A) correlates with the ability of birds to elicit a cellular immune response ( C. K. Naspitz and M. Richter, 1968 , Prog. Allergy 12 , 1–85; P. Toivanen and A. Toivanen, 1973 , J. Immunol. 111 , 1602–1603). The PHA response of splenic lymphocytes from birds infected with the avian acute leukemia viruses, myelocytomatosis virus strain 29 (MC29), avian erythroblastosis virus (AEV), avian myeloblastosis virus (AMV), or reticuloendotheliosis virus (REV-T), was completely suppressed within 1 week after virus infection of chickens by avian acute leukemia virusus. Spleen cells from chickens infected with the chronic leukemia viruses, Rous-associated viruses (RAVs) types 1 and 3, exhibited PHA responses similar to those of uninfected birds. In contrast, lymphocytes from RAV-2 infected birds had suppressed PHA responses. Acute leukemia viruses are replication-defective and are associated with replication-competent nontransforming helper viruses. When chickens were infected with helper viruses isolated from the acute leukemia virus stocks by endpoint dilution, the T-cell response to various T-cell mitogens was completely suppressed. The helper viruses, therefore, contribute to the immunosuppression which occurs during the development of avian acute leukemia. The rapid lethality of the acute leukemia viruses could be, in part, the result of the immunosuppressive activity of the associated helper viruses. Though all the acute leukemia viruses severely immunosuppress their hosts, the mechanism by which the immunosuppression was induced by these viruses differed. REV-T was the only acute leukemia virus which induced a population of splenic suppressor cells. All the viruses of the leukosis-sarcoma complex tested which had subgroup B specificity were immunosuppressive while those of subgroup A were not. These results suggest that immunosuppression by these viruses may be related to subgroup specificity.

The v-rel oncogene of avian reticuloendotheliosis virus transforms immature and mature lymphoid cells of the B cell lineage in vitro

Heavy chain gene rearrangements were analyzed in 67 independently derived reticuloendotheliosis virus (REV-T) transformed avian lymphoid cell lines. The status of the heavy chain genes in these REV-T transformed cell lines was determined, in part, by the age of the chicken whose spleen cells were transformed. Cell lines derived by the in vitro transformation of splenic lymphocytes obtained from embryos did not contain heavy chain gene Ig rearrangements. By contrast, splenic lymphocytes transformed by REV-T obtained from birds 1 week or older generally exhibited heavy chain gene rearrangements. The REV-T transformed lymphoid cell lines with heavy chain rearrangements also had light chain gene rearrangements. The Ig gene rearrangements in REV-T transformed cells were functional. The majority of the cells which had heavy chain rearrangements expressed a 2.2-kb mu transcript and synthesized and secreted IgM. An REV-T transformant was also identified which produced IgG, suggesting that v-rel can transform a terminally differentiated cell. Irrespective of their Ig chain gene status the REV-T transformed cell lines expressed variable amounts of some but not all normal B cell-specific markers and failed to express T cell markers. All the cell lines analyzed expressed the B-L (Ia-like) antigen as well as a common leukocyte antigen. Based on the expression of these surface molecules, the transformants with or without Ig gene rearrangements all appear to be committed to the B cell pathway.

The role of monovalent cation transport in Sindbis virus maturation and release

Alterations in intracellular monovalent cation concentrations in Sindbis virus-infected avian cells result, in part, from a reduction in Na+/K+ ATPase (Na+ pump) activity. Inhibition of Na+ pump activity was shown previously to temporally correlate with the appearance of viral envelope proteins on the cell surface and the release of virus particles. Cells infected with envelope-defective temperature-sensitive mutants exhibited reduced Na+ pump activity at the nonpermissive temperature, where viral particles are not released. By contrast, Na+ pump activity was not inhibited in Sindbis virus-infected cells treated with tunicamycin or with antiviral serum, which block virus maturation and release. Diuretic-sensitive transport of 86Rb+, aK+ tracer, was stimulated in cells which express virus envelope proteins, but fail to release virus particles. In these cells, the furosemide-sensitive 86Rb+ influx exhibited an increase in Vmax and was responsive to changes in the extracellular concentration of NaCl. Furosemide inhibited the rapid release of virus from low salt-inhibited cells after shift to isotonic conditions. Alterations in ion transport during alphavirus infection may, therefore, facilitate the efficient release of progeny virus particles.

Effect of tunicamycin on the development of the cytopathic effect in Sindbis virus-infected avian fibroblasts

In Sindbis virus-infected avian cells the development of the cytopathic effect is correlated with the disruption of plasma membrane function. Sindbis virus inhibits the activity of the Na+K+ATPase, a membrane-associated enzyme complex which regulates intracellular monovalent cation levels. Tunicamycin, which blocks envelope protein glycosylation, prevents inhibition of Na+K+ATPase activity and the development of morphological changes in Sindbis virus-infected cells. Although inhibition of Na+K+ATPase activity is not essential for the termination of host protein synthesis, membrane-mediated events may favor the selective translation of viral proteins. The termination of host protein synthesis does not contribute to the development of these cytopathic changes in the time frame examined. In tunicamycin-treated, Sindbis virus-infected cells, unglycosylated E1 is inserted into the plasma membrane but virus release is prevented. In productively infected cells, therefore, the inhibition of Na+K+ATPase activity and the development of the cytopathic effect may result from terminal events in virus assembly and/or virus release.

Chicken fetal antigens: Role as cell surface receptors for sindbis virus hemagglutinin

Abstract Sindbis virus attaches to and agglutinates red blood cells from fetal and developing chickens that express chicken fetal antigens (CFA). Erythrocytes from adult chickens that do not express CFA do not contain the receptor site for the Sindbis virus hemagglutinin. The attachment of Sindbis virus to erythrocytes of different avian species indicates that Sindbis virus attachment occurs only when a specific CFA determinant, CFA determinant 9, is expressed. Erythrocytes reacted with antiserum directed against CFA determinant 9 fail to attach to or to be agglutinated by Sindbis virus. These results explain the failure of Sindbis virus to agglutinate adult chicken erythrocytes and identify the receptor for the Sindbis virus hemagglutinin.

Activation of the TGF-β/Smad signaling pathway in oncogenic transformation by v-Rel

v-rel, encoded by the avian reticuloendotheliosis virus, is an acutely transforming member of the Rel/NF-κB family of transcription factors. Transformation by v-Rel is mediated by the aberrant expression of genes that are normally regulated by Rel/NF-κB. Here, we demonstrate activation of the TGF-β/Smad signaling pathway in Rel transformation. RNA and protein levels of key TGF-β and Smad family members (TGF-β2, -β3, TGF-β type II receptor, and Smad3) are upregulated in v-Rel transformed cells with little to no change in c-Rel-expressing cells. Treatment of v-Rel transformed lymphoid cells with kinase inhibitors of the TGF-β receptor dramatically reduces soft agar colony formation whereas addition of TGF-β2 further promotes transformation. Moreover, Smad3 but not Smad2, is selectively activated as the downstream mediator of TGF-β signaling. Blocking Smad3 expression or activity inhibits the oncogenic potential of v-Rel. Overall, TGF-β/Smad signaling is activated at multiple levels and is required for the transforming ability of v-Rel.

Oncogenes, protooncogenes, and signal transduction: toward a unified theory?

This paper has reviewed, in a broad sense, the potential involvement of the oncogenes and their progenitors, the protooncogenes, in signal transduction pathways. The membrane-associated oncogene products appear to be connected with the generation and/or regulation of secondary messengers, particularly those associated with Ca2+/phospholipid-dependent activation of the serine/threonine kinase protein kinase C. Activation of transmembrane receptors, either through binding their native ligand or through point mutations that lead to constitutive expression, results in the expression of their intrinsic tyrosine-specific protein kinases. In PDGF-stimulated cells, this results in the increased turnover of phosphatidylinositols and the subsequent release of IP3 (Habenicht et al., 1981; Berridge et al., 1984). This coincides with activation of a PI kinase activity (Kaplan et al., 1987). Likewise, the fms product, which is the receptor for CSF-1, induces a guanine nucleotide-dependent activation of phospholipase C (Jackowski et al., 1986). Receptor functions are potentially regulated through differential binding of ligands (as proposed with PDGF), through interactions with other receptors, and through the feedback regulation mediated by protein kinase C. PDGF stimulation leads to modulation of the EGF receptor through protein kinase C (Bowen-Pope et al., 1983; Collins et al., 1983; Davis and Czech, 1985). Similarly, the neu product becomes phosphorylated on tyrosine residues following treatment of cells with EGF, although the neu protein does not bind EGF itself (King et al., 1988; Stern and Kamps, 1988). The tyrosine kinases of the src family are not receptors themselves, although they may mediate specific receptor-generated signals. The clck product is physically and functionally associated with the T-cell receptors CD4 and CD8, and becomes active upon specific stimulation of cells expressing those markers (Veillette et al., 1988a,b). The precise physiological role of the src family products has not been established, but their kinase activity is intrinsic to that function. The v- and c-src products are hyperphosphorylated during mitosis (Chackalaparampil and Shalloway, 1988), which correlates with periods of reduced cell-to-cell adhesion and communication (Warren and Nelson, 1987; Azarnia et al., 1988). Furthermore, pp60c-src is associated with a PI kinase activity when complexed with MTAg of polyoma virus, suggesting a function in stimulating increased turnover of the phosphatidylinositols (Heber and Courtneidge, 1987; Kaplan et al., 1987).(ABSTRACT TRUNCATED AT 400 WORDS)