Induction of proliferation or transformation of neuroretina cells by the mil and myc viral oncogenes

The genome of the avian retrovirus MH2 contains, in addition to the v-myc oncogene shared with three other avian retroviruses (MC29, CMII and OK-10), a second cell-derived oncogene, v-mil (refs 1-3). Like the three other viruses, which contain only v-myc, MH2 induces mainly liver and kidney carcinomas in fowl and transforms fibroblasts and macrophages in vitro. However, MH2 and MC29 differ in their biological properties when assayed on cultures of chicken embryo neuroretina (NR) cells. Indeed, NR cells, which normally do not multiply in vitro, are induced to proliferate and become transformed upon infection with MH2, whereas infection with MC29 has no apparent effect on these cells. To analyse the functions of the two oncogenes of MH2, we isolated spontaneous and in vitro-constructed mutants of this virus and investigated their effects on NR cell multiplication and transformation. We report here that expression of v-mil is sufficient to induce NR cell proliferation, although it does not result in cell transformation. In addition, viruses expressing only the v-myc oncogene fail to induce any detectable change in NR cells. However, cooperation of the two oncogenes is required to achieve transformation of NR cells by MH2.     

The Hardy-Zuckerman 2-FeSV, a new feline retrovirus with oncogene homology to Abelson-MuLV

There is substantial evidence that oncogenes (v-onc) of acute transforming retroviruses have been acquired by transduction of cellular genes (c-onc) with retroviruses. Feline leukaemia virus (FeLV)-associated feline fibrosarcomas have proven to be extremely useful for the isolation of acute transforming retroviruses of a mammalian species. Three different v-onc genes have been identified in five acute transforming feline retroviruses. The Susan McDonough feline sarcoma virus (SMFeSV) contains the oncogene fms (ref. 4). The Snyder-Theilen (ST) and Gardner-Arnstein (GA) FeSVs contain the oncogene fes (ref. 4), which is homologous to the oncogene fps of the avian sarcoma viruses FSV, RRCII, PRCIV and 16L (refs 7, 8). The v-onc sequences of the Parodi-Irgens (PI) FeSV have recently been found to be homologous with the v-sis sequences of the simian sarcoma virus. We report here the isolation of another acute transforming feline retrovirus from a naturally occurring feline fibrosarcoma, designated the Hardy-Zuckerman 2 feline sarcoma virus (HZ2-FeSV) and demonstrate that the HZ2-FeSV and Abelson murine leukaemia virus (A-MuLV) have homologous oncogenes.     

Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus

The complete nucleotide sequence of the transforming gene of a mouse sarcoma virus has been determined. It codes for a protein of 374 amino acids. The nucleotide sequence of the junctions between a murine leukaemia virus and cellular sequences leading to the formation of the viral transforming gene have also been elucidated. The viral transforming sequence and its cellular homologue share an uninterrupted stretch of 1,159 nucleotides, with few base substitutions. The predicted amino acid sequence of the mouse sarcoma virus transforming gene was found to share considerable homology with the proposed amino acid sequence of the avian sarcoma virus oncogene (src) product.     

A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family

A new acute transforming feline retrovirus, the Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), has been isolated from a feline fibrosarcoma. The viral genome of HZ4-FeSV contains a new oncogene designated v-kit, has the structure 5' delta gag-kit-delta pol-delta env 3' and specifies a gag-kit polyprotein of relative molecular mass 80,000. The predicted kit amino-acid sequence displays partial homology with tyrosine-specific protein kinase oncogenes. HZ4-FeSV appears to have been generated by transduction of feline c-kit sequences with feline leukaemia virus.     

Tumorigenic transformation of mammalian cells induced by a normal human gene homologous to the oncogene of Harvey murine sarcoma virus

A normal human gene homologous to the p21 ras oncogene of Harvey murine sarcoma virus induced oncogenic transformation and high p21 ras levels in murine fibroblasts when this gene was ligated to a control element (the long terminal repeat) from a murine or feline retrovirus. These results indicate that high levels of a gene product encoded by a normal human oncogene can induce tumorigenic transformation.     

Mature murine B lymphocytes immortalized by Kirsten sarcoma virus

Clonal, antigen-specific, functionally responsive cell populations have proved critical for the analysis of the activation and regulation of lymphocytes. Such studies with B lymphocytes, the precursors of antibody-secreting cells, are hampered by the difficulty in generating phenotypically mature, antigen-reactive lines from defined cell populations. One method is to use acutely transforming retroviruses, which can transform B-lineage lymphocytes in vitro. However, Abelson murine leukaemia virus (A-MuLV) infection of murine bone marrow cells in vitro yields mostly immature B-cell lines, and infection of murine bone marrow cells with murine sarcoma viruses carrying ras related genes produces only immature lymphoid cell lines. Retroviruses which contain ras can immortalize nonlymphoid cells without causing loss of mature phenotypic characteristics. We used ras-containing Kirsten sarcoma virus (KiSV) pseudotyped with an amphotropic MuLV helper virus, to infect a purified population of mature, hapten-binding murine splenic B lymphocytes, aiming to generate mature B-cell lines to use as models for the study of B-cell growth and differentiation physiology. Immortalized B-cell lines which retain the same mature phenotype as the starting population, including hapten-specific binding, were produced. This is the first demonstration of a method for immortalizing selected antigen-binding B lymphocytes, and the first example of immortalization of mature B cells in vitro with an acutely transforming ras-containing retrovirus.     

Purified EGF receptor-kinase interacts specifically with antibodies to Rous sarcoma virus transforming protein

Transformation by several RNA tumour viruses seems to be mediated by virally coded protein kinases which specifically phosphorylate tyrosine. A tyrosine-specific protein kinase also seems to be involved in the mitogenic action of epidermal growth factor (EGF). This EGF-stimulated kinase activity is closely associated with the EGF receptor, with which it copurifies during EGF-affinity chromatography. Because both the virus- and EGF-stimulated tyrosine kinases may be involved in stimulation of cell growth, and because the viral kinases may be antigenically related to normal cell proteins, we examined the interaction of antibodies to viral tyrosine kinases with the affinity-purified EGF receptor-kinase preparation. We report here that the receptor-kinase specifically phosphorylates antibodies directed against the transforming protein kinase pp60src of Rous sarcoma virus. However, none of these antibodies, including those which cross-react with the normal cellular homologue of pp60src (pp60sarc), precipitate the receptor-kinase. These results suggest that the EGF receptor-kinase is related to, but probably not identical with, pp60sarc.     

The p21 ras C-terminus is required for transformation and membrane association

The Harvey murine sarcoma virus (Ha-MuSV) transforming gene, v-rasH, encodes a 21,000 molecular weight protein (p21) that is closely related to the p21 proteins encoded by the cellular transforming genes of the ras gene family. The primary translation product (prop21), which is found in the cytosol, undergoes posttranslational modification and the mature protein subsequently becomes associated with the inner surface of the plasma membrane and binds lipid tightly. The p21 proteins have the capacity to bind guanine nucleotides non-covalently in vitro. To assess the biological relevance of these biochemical features of the protein, we have now studied a series of deletion mutants located at or near the C-terminus of the viral p21 protein. Our tissue culture studies indicate that amino acids located at or near the C-terminus are required for cellular transformation, membrane association and lipid binding.     

Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites

p60src, the transforming protein of Rous sarcoma virus (RSV), is a protein kinase that has a strict specificity for tyrosine. The phosphorylation of cellular proteins by p60src (ref. 4) results in transformation. Recently, Barker and Dayhoff discovered that residues 259-485 of p60src have 22% sequence identity with residues 33-258 of the catalytic subunit of cyclic AMP-dependent protein kinase, an enzyme that has a specificity for serine. Because it was necessary to introduce eight gaps to align the two proteins, the question remained as to whether this apparent homology reflected a common evolutionary origin. We demonstrate here that the ATP analogue p-fluorosulphonylbenzoyl 5'-adenosine (FSBA) inactivates the tyrosine protein kinase activity of p60src by reacting with lysine 295. When aligned for maximum sequence identity, lysine 295 of p60src and the lysine in the catalytic subunit which also reacts specifically with FSBA are superimposed precisely. This functional homology is strong evidence that the protein kinases, irrespective of amino acid substrate specificity, comprise a single divergent gene family.     

Avian sarcoma virus Y73 genome sequence and structural similarity of its transforming gene product to that of Rous sarcoma virus

From the complete nucleotide sequence of the genome of the avian sarcoma virus Y73, we have predicted amino acid sequence of p90 gag-yes, the product of the transforming gene. Contrary to previous evidence from molecular hybridization studies p90 gag-yes was found to have much homology with the transforming gene product p60 src of Rous sarcoma virus, suggesting that the cellular counterparts of the two (c-yes and c-src) originated from a common prototype sequence.     

A novel viral oncogene with structural similarity to phospholipase C

Numerous oncogenes have been isolated from acutely transforming retroviruses. To date, the products of these viral oncogenes have been protein kinases, nuclear proteins, growth factors, or GTP-binding proteins. We have cloned the previously uncharacterized avian sarcoma virus CT10 and sequenced its genome. This virus encodes a protein, p47gag-crk, that has blocks of sequence similarity to the amino-terminal, non-catalytic region of the non-receptor class of tyrosine kinases. In addition, the structure of p47gag-crk has striking similarity to a 180-amino acid region of bovine brain phospholipase C. Biochemical data suggest that p47gag-crk activates one or several endogenous tyrosine kinases.     

Homology between phosphotyrosine acceptor site of human c-abl and viral oncogene products

The human homologues of several independent viral oncogenes, each of which encodes tyrosine-specific protein kinases, have been identified. Of these, three (v-src, v-yes and v-fes/fps) are known to exhibit considerable sequence homology, particularly in the regions of their phosphorylation acceptor sites. In the present study, sequences encoding the tyrosine phosphorylation acceptor sites of the Abelson murine leukaemia virus oncogene, v-abl, and its human cellular homologue, c-abl, have been identified and their nucleic acid sequences determined. Our results establish extensive homology between this region of c-abl and acceptor domains of the v-src, v-yes and v-fes/fps family of viral oncogenes, as well as more distant relatedness to the catalytic chain of the mammalian cyclic AMP-dependent protein kinase. These findings suggest that, of the homologues of retroviral oncogenes with tyrosine protein kinase activity examined to date, all were probably derived from a common progenitor and may represent members of a diverse family of cellular protein kinases.     

Transmission of the polyoma virus middle T gene as the oncogene of a murine retrovirus

Polyoma virus is a papovavirus that productively infects mouse cells. In cells of other species, such as rat cells, polyoma virus is virtually unable to replicate, and a small proportion of infected cells become stably transformed. The ability of polyoma virus to transform infected cells is determined by genes that encode the large, middle and small T antigens and which are found in the early region of the virus genome. We have inserted the transforming region of polyoma virus into a murine leukaemia virus (MLV) vector, to generate a replication-defective transforming retrovirus which for the first time allows efficient transformation of mouse cells by the polyoma virus middle T gene. During the life cycle of this recombinant virus the intervening sequence present in the original polyoma virus middle T gene was removed. The recombinant virus that we have constructed is analogous to other acutely transforming retroviruses, and demonstrates that the polyoma middle T gene is a dominant transforming oncogene.     

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.     

Guanine nucleotide-binding and autophosphorylating activities associated with the p21src protein of Harvey murine sarcoma virus

The purified p21src protein of Harvey sarcoma virus shows a guanine nucleotide-binding activity and, in addition, at elevated temperature an autophosphorylating activity at a threonine residue using as phosphoryl donor GTP or dGTP but not ATP or dATP. These biochemical activities are unique among those associated with transforming proteins of RNA-containing or DNA-containing tumour viruses.     

Abrogation of IL-3 and IL-2 dependence by recombinant murine retroviruses expressing v-myc oncogenes

Several oncogenes are thought to cause transformation by affecting the signal transmission pathway of growth factors. One example is the induction of c-myc, the cellular homologue of the avian transforming oncogene v-myc, by platelet-derived growth factor (PDGF) among a set of genes associated with competence induction in fibroblasts. Another of the competence genes, r-fos, has been shown to be related to v-fos, the transforming gene of the FBJ sarcoma virus. In addition, PDGF induces c-fos, the cellular homologue of v-fos. The importance of c-myc induction is suggested by the observation that c-myc, under the control of a glucocorticoid regulator, can partially relieve the requirement of fibroblasts for PDGF. We have examined the effects of oncogenes on haematopoietic/lymphoid cell differentiation, immortalization and factor dependence for growth. Here we report the effects of recombinant murine retroviruses capable of expressing the avian v-myc. With interleukin-3 (IL-3)- or interleukin-2 (IL-2)-dependent cells, the viruses abrogated the requirement for growth factors and suppressed c-myc expression.     

onc sequences (v-fes) of Snyder-Theilen feline sarcoma virus are derived from noncontiguous regions of a cat cellular gene (c-fes)

Type C sarcoma viruses are genetic recombinants containing portions of replication-competent helper viruses linked to sarcoma virus-specific sequences (generically designated onc genes) which are thought to be required for acute fibroblast transformation. The onc elements of different avian and mammalian sarcoma viral isolates are each homologous to subsets of cellular DNA sequences which have no well-defined role in normal cells. Because of the lack of significant homology between helper viral genes and cellular onc sequences, the recombinational mechanisms which facilitate the formation of sarcoma viral genomes remain unclear. In Moloney murine sarcoma virus, viral onc (or v-mos) and cellular onc (or c-mos) sequences exhibit complete and uninterrupted homology as determined by heteroduplex and restriction enzyme analyses of molecularly cloned DNA. By contrast, the cellular counterparts of the onc elements of Rous sarcoma virus (G. Cooper and R. Parker, personal communication), avian erythroblastosis virus (B. Vennstrom, personal communication), Abelson leukaemia virus (D. Baltimore, personal communication), Harvey sarcoma virus (E. Scolnick, personal communication) and simian sarcoma virus (R. Gallo, personal communication) are now known to contain intervening sequences which do not appear in the respective viral genomes. Here we report the use of the Southern blot technique to examine cat cellular DNA sequences (c-fes) homologous to the onc gene (v-fes) of Snyder-Theilen feline sarcoma virus (ST-FeSV). We used cloned DNA 'probes' containing defined portions of the ST-FeSV genome to show that v-fes sequences originate from at least four noncontiguous sequences in cat cellular DNA, separated from each other by intervening sequences.     

Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor

Retrovirus protease is an enzyme that cleaves gag and gag-pol precursor polyproteins into the functional proteins of mature virus particles. The correct processing of precursor polyproteins is necessary for the infectivity of virus particles: in vitro mutagenesis which introduces deletions into the murine leukaemia virus genome produces a protease-defective virus of immature core form and lacking infectivity. A therapeutic drug effective against disease caused by retrovirus proliferation could likewise interfere with virus maturation. The primary structure has so far been determined for the protease of avian myeloblastosis virus, and of murine, feline and bovine leukaemia viruses. Amino acid sequencing of the retrovirus proteases, either after their purification or from prediction from the nucleotide sequence, shows that they possess the Asp-Thr-Gly sequence characteristic of the aspartyl proteinases. In this report we show that retrovirus proteases belong to the aspartyl proteinase group and demonstrate an inhibition by the aspartyl proteinase-specific inhibitor, pepstatin A, on the activity of bovine leukaemia, Moloney murine leukaemia and human T-cell leukaemia virus proteases.