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.     

Isolation of a murine retrovirus with a temperature-sensitive src gene

A murine retrovirus containing the src gene of avian sarcoma virus ts LA31A was generated. In contrast to the avian sarcoma virus, our recombinant murine retrovirus can efficiently infect mammalian cells. Transformation of NIH3T3 cells by the recombinant murine retrovirus is temperature sensitive. At the permissive temperature of 34 degrees, cells form foci of rounded cells. At the nonpermissive temperature of 39 degrees, the infected cells remain flat and exhibit contact inhibition. No disease was observed following infection of newborn NFS/n mice with the ts mutant virus. In contrast, infection of newborn NFS/n mice with a recombinant murine retrovirus containing the wild-type src gene causes fibrosarcomas and hepatosplenomegaly.     

Stable expression of the avian retroviral oncoprotein v-Rel in avian, mouse, and dog cell lines

Overexpression of the retroviral oncoprotein v-Rel can rapidly transform and immortalize a variety of avian cells in culture. However, mammalian models for v-Rel-mediated oncogenesis have been compromised by the fact that high-level expression of v-Rel has been reported to be toxic in many mammalian cell types, including mouse 3T3 cells, Rat-1 cells, and mouse bone marrow cells. In this article, we demonstrate that 3T3 cells can support expression of v-Rel for at least 24 days when infected with a mouse stem cell virus (MSCV) retroviral vector containing v-rel. In retrovirus-infected 3T3 cells, v-Rel is located in the nucleus and can bind to DNA, but does not transform the cells. On the other hand, 3T3 and Rat-2 cells do not express v-Rel after stable transfection with a pcDNA-based v-Rel expression vector. We also show that infection of the IL3-dependent mouse B cell line BaF3 with the MSCV-v-rel vector results in expression of v-Rel, but does not convert these cells to growth factor independence. In contrast to 3T3 cells, the dog osteosarcoma D17 cell line can support a high level of v-Rel expression, after either transfection or infection with a retroviral vector. That is, v-Rel can be stably expressed as a nuclear, DNA-binding protein in D17 cells to approximately the same level as in chicken embryo fibroblasts. These results suggest that the restriction to v-Rel expression in rodent fibroblasts is generally absent in D17 cells and that the type of v-rel expression vector determines whether 3T3 cells can support stable expression of v-Rel. The findings reported here are an essential first step in the development of mammalian systems to study Rel-mediated oncogenesis.     

Avian cells expressing the murine Mx1 protein are resistant to influenza virus infection

The cDNA encoding the murine Mx1 protein, a mediator of resistance to influenza virus, was inserted into a replication-competent avian retroviral vector in either the sense (referred to as Mx+) or the antisense (referred to as Mx-) orientation relative to the viral structural genes. Both vectors produced virus retaining the Mx insert (Mx recombinant viruses referred to as Mx+ and Mx-) following transfection into chicken embryo fibroblasts (CEF). Mx protein of the appropriate size and nuclear localization was expressed only in CEF cells infected with the Mx+ virus. Mx expression was observed in all Mx(+)-infected cells and was stable during long-term culture. Cells infected with the Mx+ virus were resistant to infection by human influenza A/WSN/33 (H1N1) and avian influenza viruses A/Turkey/Wisconsin/68 (H5N9) and A/Turkey/Massachusetts/65 (H6N2), but were susceptible to infection by the enveloped RNA viruses Sindbis and vesicular stomatitis virus (VSV). Normal CEF and cells infected with the Mx virus were susceptible to influenza A, Sindbis, and VSV. The synthesis of influenza proteins, especially the larger polymerase and hemagglutinin proteins, was reduced in Mx+ retrovirus-infected cells superinfected by influenza A.     

Deposition of retrovirus associated antigens (p30 and gp70) on cell membranes of feline and murine leukaemia virus infected cells.

A quantitative estimation of retrovirus associated cell membrane antigens of murine and feline cells infected with their respective type C leukosis virus is presented. Using a radio-immune assay with three broadly reactive antisera, the minimum estimated number of retrovirus associated antigenic determinants on YAC [Moloney leukaemia virus (MuLV) infected murine] and FL-74 [feline leukaemia virus (FeLV) infected feline] cells was 1.3 x 10(6) and 1.6 x10(6) determinants per cell respectively. The virus structural proteins p27-30 and gp70 were detected by three component specific antisera on murine and feline cell surfaces in amounts which varied between cell isolates. MuLV infected cells produced as many as 1.9 x 10(5) p30 antigenic determinants and 7.5 x 10(5) gp70 determinants on infected cells. FeLV infected cells (FL-74) expressed 5.6 x 10(5) p27 and 7.5 x 10(5) gp70 antigenic determinants per single cell surface. The major core protein (p27-30) and the major envelope glycoprotein (gp70) antigens are sufficiently physically separated on cell surfaces so that binding of either of the membrane antigens with component specific antibodies does not interfere with binding of antibodies specific for the other. Despite the expression of interspecies determinants for p30, gp70, and other retrovirus associated antigens detected by antibody procedures, interspecies determinants of cell mediated immunity could not be demonstrated in immune mice bearing Moloney sarcoma virus (MSV) induced tumours. Furthermore, xenogeneic immunization of mice with FL-74 cells failed to protect mice against the growth of MSV induced lymphoma or sarcoma.     

The transforming protein of avian reticuloendotheliosis virus is a soluble cytoplasmic protein which is associated with a protein kinase activity

We have identified the product (p57v-rel) of the transforming gene, v-rel, of avian reticuloendotheliosis virus (REV-T) using antisera generated against nonoverlapping sequences representing the middle and carboxy-terminal regions of the v-rel protein expressed in Escherichia coli (N.K. Herzog and H.R. Bose, Jr., 1986, Proc. Natl. Acad. Sci. USA 83, 812-816). The amino-terminal region of the v-rel protein was also expressed in E. coli and used to generate antisera. The immunoglobulin-enriched fractions of these antisera were used to determine the subcellular location of p57v-rel in REV-T transformed lymphoid cells. Cells were fractionated into nuclear, mitochondrial, microsomal, and cytoplasmic fractions. The majority of p57v-rel was found in the cytoplasm. Examination of REV-T transformed lymphoid cells labeled with 32Pi revealed that the majority of the phosphorylated form of the v-rel protein was also found in the cytoplasm. Indirect immunofluorescence of REV-T transformed cells gave a diffuse cytoplasmic pattern indicating that p57v-rel was not associated with any discrete cellular organelle. The distribution of p57v-rel was similar in REV-T transformed lymphoid cells labeled with [35S]methionine for short and long periods of time, suggesting that p57v-rel is a soluble cytoplasmic protein throughout its lifetime. The v-rel protein was phosphorylated when immune complexes precipitated from transformed cells with the immunoglobulin fractions obtained from antisera against the amino-terminal, middle, and carboxy-terminal regions of v-rel were incubated with [gamma-32P]ATP and Mn2+. The phosphorylation of p57v-rel in the in vitro immune complex kinase assay was inhibited when the immunoglobulin-enriched fraction of these antisera was preincubated with the homologous v-rel fusion proteins. Preincubation with heterologous proteins did not block the phosphorylation of p57v-rel. These observations suggest that p57v-rel is associated with a protein kinase activity. Most of the kinase activity was found in the soluble cytoplasmic fraction of transformed cells. The transforming protein encoded by v-rel is a relatively stable protein with a half-life of approximately 7 to 8 hr in transformed lymphoid cells.     

Ecotropic murine leukemia virus envelope protein affects interaction of cationic amino acid transporter 1 with clathrin adaptor protein complexes, leading to receptor downregulation

Mouse cationic amino acid transporter 1 (mCAT1) serves as the receptor for ecotropic murine leukemia virus (eMuLV). It has been shown that mCAT1 is expressed on the basolateral surface of polarized epithelial MDCK cells. However, little is known about the mechanisms involved in the intracellular trafficking of mCAT1. Using the green fluorescent protein-tagged mCAT1 expressed in MDCK cells, we report here that mCAT1 is physically associated with clathrin adaptor protein complex 1 (AP-1) implicated in protein trafficking from trans-Golgi network (TGN) to the basolateral surface. When the cells were infected with eMuLV, reduction of cell surface mCAT1, as well as a concomitant decrease in mCAT1-AP-1 association, was observed while association of mCAT1 with AP-3 involved in the TGN-to-lysosome trafficking was increased. Similar results were obtained when eMuLV envelope protein alone was expressed. The results may provide useful insights into the mechanism by which a simple retrovirus downregulates its receptor.     

Avian oncovirus proteins expressed on the surface of infected cells

Lactoperoxidase-catalyzed iodination and anti-AMV immunoprecipitation showed that both chicken and duck fibroblasts infected with a sarcoma virus (Rous sarcoma virus PrC) or a leukemia virus (avian myeloblastosis virus; AMV) had on their surface a protein of approximately 120 kilodaltons molecular weight (120K), as well as envelope glycoprotein precursors of 90–92 kd. Uninfected chicken fibroblasts of the gs^?, chf^? phenotype had much lower, but detectable amounts of surface 120K, whereas uninfected duck fibroblasts did not have any, suggesting a relationship between surface 120K and expression of chicken virus information in the cell. 120K is a glycoprotein, since it could be labeled with [³H]mannose and contained a component that bound to a concanavalin A affinity column. The 120K protein was characterized by tryptic fingerprinting after reiodination with chloramine-T. Total and Con A-selected 120K from infected chicken cells and total 120K from infected duck cells had essentially identical fingerprints. Moreover, they were extensively related to the iodinated fingerprint of Pr76^gag, the intracellular precursor of viral core proteins. These results indicate that expression on the cell surface of glycosylated forms of gag polyproteins occurs also in avian oncornavirus infections, similarly to findings in the murine leukemia virus system.     

Mouse xenotropic viruses can inhibit cell transformation

Mammalian type C retroviruses can be detected by their induction of foci of cell transformation in S+I - cells. We have noted that certain subtypes of the mouse xenotropic type C retrovirus (MuLV) inhibit this cell alteration. This inhibition, associated with intact virions, is irreversible and gives the infected cells a phenotype of uninfected cells. In comparison to the transformed cells, the inhibited cultures showed primarily a decrease in murine sarcoma virus (MSV) progeny production concomitant with a reduction in the MSV mos RNA expression. No difference in beta-actin RNA production was observed between the inhibited and transformed cultures. This selective effect of mouse xenotropic MuLV on MSV and mos RNA production in these cells focuses attention on the mechanism of transformation in this system.     

The v-rel oncogene product is complexed with cellular proteins including its proto-oncogene product and heat shock protein 70

The oncogene product, pp59v-rel, of avian reticuloendotheliosis virus (REV-T) is complexed in the cytosol of REV-T transformed lymphoid cells with cellular proteins. Monoclonal antibodies and antisera directed against different regions of pp59v-rel coimmunoprecipitate five cellular proteins (p124, p115, p75, p70, and p40) in addition to pp59v-rel. Cellular proteins with the same apparent molecular mass also copurify with pp59v-rel during sequential Sephacryl S200 and immunoaffinity chromatography. Antisera directed against the most abundant cellular protein in the complex, pp40, coimmunoprecipitate pp59v-rel and several cellular proteins with the same apparent molecular mass. The 75-kDa protein in the pp59v-rel complex is the product of c-rel proto-oncogene and is weakly phosphorylated. In MSB-1 cells this protein is not detectably phosphorylated or associated with cellular proteins. The 70-kDa protein in the pp59v-rel containing cytosolic complex is the constitutive form of avian heat shock protein 70 (HSC70). The p70 protein coimmunoprecipitates and copurifies with pp59v-rel using antisera directed against pp59v-rel and coimmunoprecipitates with antisera specific for pp40. The p70 isolated from immune complexes containing pp59v-rel shares V8 protease fragments with HSC70.     

Avian hemangioma retrovirus induces cell proliferation via the envelope (env) gene

Several years ago, a field strain retrovirus, avian hemangioma virus (AHV), was isolated from hemangioma tumors in layer hens. Sequence analysis indicated that the AHV genome contains the three prototypic retroviral genes, gag, pol, and env, and is devoid of an oncogene. In cultured endothelial cells, however, AHV induced a significant cytopathic effect through a typical apoptotic cascade. We now demonstrate that AHV also induces cell proliferation and anchorage-independent growth of BSC-1 epithelial cells and NIH-3T3 fibroblasts. This was shown by measurements of (1) cell viability, (2) DNA synthesis, (3) flow cytometry analysis of the cell DNA content, and (4) clonogenic efficiency of the infected cells. Anchorage-independent cell growth was demonstrated by colony formation in soft agar. Moreover, the AHV env gene was cloned into a MuLV-based retroviral vector, and infection of NIH-3T3 cells with this vector induced cell proliferation as well as clonogenic growth. These results suggest that AHV, which is devoid of an oncogene, is a pleiotropic activator capable of inducing either apoptosis or cellular proliferation, depending on the infected cell type.     

Expression of immunoglobulin genes in the avian embryo bone marrow revealed by retroviral transformation.

Analysis of the early stages of avian B lymphocyte differentiation has been hampered by the low frequency of extra-bursal B lineage cells in sites of hematopoiesis. Consequently, little is known about B lineage precursors prior to their migration into the bursa of Fabricius. Colonization of the bursa typically occurs between about days 8 and 14 of embryonic (e) development, although cells which can colonize the bursa, functionally defined as pre-bursal stem cells, can be demonstrated in embryo bone marrow up until about the time of hatch. As a novel approach to analyzing early stages of avian B lymphocyte development, we show here that transformed B lineage cells can be derived from chick embryo bone marrow after infection in vitro with the replication-defective retrovirus REV-T produced in the context of the non-cytopathic CSV helper virus. Thus, exposure of day 14e-15e chick embryo bone marrow cells to REV-T (CSV) results in the generation of transformed, polyclonal lines of cells. From these lines, cells expressing cell surface immunoglobulin were readily isolated by flow cytometric cell sorting and single cell cloning. Analysis of the phenotype of REV-T(CSV)-transformed clones with a panel of monoclonal antibody reagents demonstrated that transformation by v-rel likely leads to marked changes in cell surface antigen expression. Nonetheless, clones expressing cell surface immunoglobulin expressed apparently normal mRNA for immunoglobulin mu and light chain and contained apparently normal immunoglobulin heavy and light chain gene rearrangements. Furthermore, no evidence for chromosomal deletions or aberrations of the Ig loci was detected among either sIg+ or sIg- REV-T(CSV)-transformed clones.     

Recovery of a rare clone from a population of unstable retroviral vector-expressing mammalian cells using a new RNA extraction and slot-blot protocol

Although a useful and important method of gene transfer, retroviral vectors can be genetically unstable. In the course of experiments using DOEJS, a retroviral vector able to confer expression of a H-ras oncogene and a neomycin resistance gene (neo) on mammalian cells (Compere et al., 1989), it was found that the vast majority of infected rat embryo fibroblasts, recovered on the basis of neo activity (i.e., G418 resistance), did not express ras mRNA. It was subsequently observed that most cells in the ψ2 cell line used to propagate DOEJS failed to produce virus capable of expressing both ras and neo in primary rat embryo fibroblasts. A simplified RNA extraction and slot-blot technique was developed to screen mRNA from several hundred fibroblast clones and, in doing so, infected fibroblast clones producing both neo and ras mRNA were identified at low frequency. The DOEJS/ψ2 packaging line was subsequently subcloned and individual clones screened for their ability to confer appropriate gene expression on target cells. Subclone DOEJS/ψ2-B6 was eventually isolated after screening 24 DOEJS subclones and 240 infected rat embryo fibroblast colonies. DOEJS/ψ2-B6 was shown to induce reliably phenotypic transformation, G418 resistance, and ras and neo mRNA expression in primary rat embryo fibroblasts. The RNA extraction and screening procedure was thus useful for recovering an infrequent subclone producing a retrovirus with the original properties.     

Effect of interferon on the growth of Chlamydia trachomatis in mouse fibroblasts (L cells).

The effect of murine interferon on the growth of the lymphogranuloma venereum biotype of Chlamydia trachomatis (strain 440L) in murine fibroblasts (L cells) was examined. Treatment of infected cell cultures with interferon caused a reduction in the number of inclusion-bearing cells as seen by light and electron microscopy and a decrease in yields of chlamydiae as determined by infectivity assays. Interferon also inhibited cycloheximide-resistant (chlamydia-specific) protein synthesis in infected cells. The interferon effect was dose dependent, with 80 to 90% inhibition occurring at concentrations of greater than 200 IU/ml. The inhibitory effect was neutralized by anti-murine interferon globulin. Interferon did not inactivate extracellular chlamydiae, and both host cell RNA and protein synthesis were required for the development of the interferon-induced antichlamydial state. Inhibition of chlamydial growth by interferon was demonstrable in cells treated 18 h before infection or up to 4 h after infection. Cells infected after interferon was removed exhibited an antichlamydial activity decline which was complete by 30 h after interferon removal. We show that interferon treatment did not affect either entry of chlamydiae into host cells or chlamydial conversion to reticulate bodies but rather caused a reduction in the rate of reticulate body replication.