RNA Tumor Viruses Research Articles

Transformation-related proteins associated with kirsten sarcoma virus

The protein composition of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus (KiSV) has been characterized. These noninfectious particles contain KiSV RNA, reverse transcriptase, and the major core protein, p28, of the helper woolly monkey leukemia virus. Instead of the usual complement of helper virus proteins, these defective particles include several polypeptides previously not identified in RNA tumor viruses. Two proteins, gp110 and gp55, of 110,000 and 55,000 daltons, were glycoproteins. The third protein, p20, of 20,000 daltons, was a phosphoprotein, as was gp110. We show by immunoprecipitation that the two glycoproteins were common to a variety of different rodent viruses. In contrast, the 20,000-dalton protein appeared to be specific to the KiSV. All three proteins were associated with cell membranes. Analysis of the processing and release of these proteins from rat cells revealed that gp55 had a markedly short half-life of 20 min whereas gp110 and p20 had long half-lives similar to those of most cell membrane proteins. The presence of gp110 and p20 in particles was dependent upon transformation and independent of helper virus infection. Both gp110 and p20, but not gp55, showed a variety of alterations following transformation of cells by Kirsten sarcoma virus. The amount of gp110 in transformed cells increased both in cell plasma membranes and in the medium. In addition, gp110 became phosphorylated and accessible to lactoperoxidase radioiodination. The alterations of gp110 with respect to the cell membrane were independent of the transforming agent since we observed the same results in BALB/3T3 cells transformed by an RNA virus (Kirsten), a DNA virus (SV40), and a chemical carcinogen (methylcholanthrene). Thus, gp110 is a transformation-related cell surface marker. P20 showed similar changes upon transformation: The amount of p20 increased dramatically in membranes; in addition, p20 appeared as a double band, became phosphorylated, and was shed into the medium. However, p20 was not accessible to lactoperoxidase radiodination. The immunological specificity and many of the biochemical cheracteristics of this protein were similar to the properties ascribed to the Kirsten sarcoma virus src gene product.

Human leukemia viruses? RNA tumor viruses, human malignancies, and concepts of viral carcinogenesis.

For an elucidation of the definitive role of retroviruses in human carcinogenesis the expression of viral parameters during the course of induced malignant diseases has to be studied. For obvious reasons these studies can only be carried out in animal systems. On the basis of such studies the human data will have to be reevaluated as to what the function of the viruses for the course of malignant diseases might be. In addition, with the development of more sensitive techniques for the detection of viral footprints, such as genetic engineering, the significance of retroviruses with regard to human neoplasms will have to be reevaluated. Finally, in view of the well-known role of many factors in carcinogenesis and the probable multifactorial etiology of human cancer, the concept of cocarcinogenesis and the role of cofactors other than viruses, such as radiation and chemicals, aging, hormones, graft vs. host reaction, environmental factors, etc., will have to be carefully considered.

Malignancies of metastatic murine lymphosarcoma cell lines and clones correlate with decreased cell surface display of RNA tumor virus envelope glycoprotein gp70.

Variant sublines of the murine lymphosarcoma RAW117 have been derived by sequential cycles of intravenous inoculation of cells and harvesting of solid liver tumors in syngeneic BALB/c mice [Brunson K. W. & Nicolson, G. L. (1978) J. Natl. Cancer Inst. 61, 1499-1503] and also by sequential removal of lectin-reactive cells via repeated adsorption on immobilized-lectins [Reading, C. L. Belloni, P. N. & Nicolson, G. L. (1980) J. Natl. Cancer Inst. 64, 1241-1249]. These cell sublines and their clones were analyzed for abilities to form gross liver tumor metastases after injection intravenously or subcutaneously into syngeneic mice, and this response was related to certain cell surface properties including quantities of viral antigens and lectin-binding sites, exposure of specific cell surface proteins, and quantities of cell surface glycoproteins visualized in gels with 125I-labeled lectins or antibodies. Consistent differences were obtained between RAW117 sublines of low and high malignancy with respect to the amounts or exposures of cell surface glycoprotein components of Mr approximately 70,000 or 69,000 and 71,000, depending on the gel system. Competition radioimmunoassays for RNA tumor virus antigens in the RAW117 lines and clones indicated the presence of Moloney murine leukemic virus antigens gp70, p30, and p12. Enhanced malignancy and metastasis to liver was accompanied by decreases in the cellular contents of viral antigens and loss of gp70 cell surface exposure. Analysis of several clones obtained from sublines selected in vivo and in vitro for high or low malignancy confirmed the inverse relationship between metastasis and expression of gp70 in this system.

The Molecular Biology of RNA Tumor Viruses: A Physician's Guide

The possibility that certain tumor viruses are simply laboratory artifacts makes them in some respects more interesting rather than less, because it means that they may be offering us a way of isolating and studying the genes responsible for the cancerous state. That seems to be the real justification for putting so much effort into investigating the tumor viruses, not the vague hope that human cancer will turn out to be a virus disease.1 RNA tumor viruses (or "retroviruses") are proved agents of oncogenesis in certain species.2 It is therefore not surprising that in recent years there has been a . . .

Activation of cellular genes by avian RNA tumor viruses.

We demonstrated previously that chicken embryo fibroblasts accumulate approximately 100 copies of embryonic globin RNA after transformation by Rous sarcoma virus. Here we demonstrate that the globin gene in chicken embryo fibroblasts is activated by infection with two other oncogenic retroviruses, avian erythroblastosis virus and strain MC-29 of avian myeloblastosis virus, which contain transforming genes unrelated in nucleotide sequence content to each other or to the Rous sarcoma virus src gene. In addition, we have measured the genetic complexity of transformation by using established techniques for determining the number of different RNA sequences in specific populations of cells. Our results indicate that transformation of chicken embryo fibroblasts by Rous sarcoma virus results in the accumulation of RNA from approximately 1000 average-sized new transcription units.

Prevalent deficiency in tumor cells of cycloheximide-induced cycle arrest.

Mammalian cell growth is regulated by a process that is completed at a restriction point in the late G1 part of the cycle. This process is highly sensitive to serum concentration and to moderate inhibition of protein synthesis by cycloheximide (CHM) or other agents. We have proposed that a cell must accumulate a labile protein in a critical amount before events related to its DNA synthesis can start. The accumulation of this protein requires conditions suitable for growth, including sufficient amounts of serum-derived factors. An important criterion for attributing a major role to such a regulatory mechanism is that cells whose growth control is modified--e.g., by mutation--should be defective in this process. Cells of this kind are produced by tumorigenic transformation. We show here that mouse 3T3 cells, human fibroblasts, and Chinese hamster CHEF/18 cells have stringent G1 growth control by CHM. In contrast, tumorigenic lines obtained from these cells by transformation with varius agents (DNA tumor virus, RNA tumor virus, chemical carcinogens) or spontaneously all showed relaxed growth control under the influence of CHM. In these lines, growth control was relaxed to different degrees; some lines were held in G1 by a combination of low serum concentration and CHM, but others were not. Serum concentration showed a synergistic effect with CHM. Low serum concentrations did not limit growth only by affecting the rate of protein synthesis. The labile-protein mechanism is likely to be basic to growth control by serum factors. Transformed tumorigenic cells in general may have relaxed this mechanism.

Cytochalasin B-induced multinucleation of human tumor and normal cell cultures

Twelve human cell cultures derived from tumors, normal tissues, and derivative cultures transformed by either a RNA tumor virus or chemical carcinogen were examined for their response to cytochalasin B (CB) and the expression of this marker was correlated with growth in soft agar and saturation density in monolayer culture. Cell lines derived from carcinoma of the bladder (T24 and RT4), kidney (Caki-1), prostate (DU 145), and breast (MCF-7) multinucleated when grown in CB-supplemented medium, whereas cell cultures derived from benign bladder epithelium (HCV-29), and normal kidney (Flow 4000) and skin (GM10) remained binucleate under comparable conditions. Human osteosarcoma (HOS) cells transformed by murine sarcoma virus (MSV) or a chemical carcinogen extensively multinucleated in response to CB treatment, relative to a morphological revertant of MSV-transformed HOS and parental HOS cells. CB-induced multinucleation was consistently correlated with the ability of cells to form colonies in soft agar but inconsistently correlated with growth to high saturation densities. These results demonstrate a differential response to CB by normal and transformed human cells and that CB-induced multinucleation provides a convenient and useful in vitro marker for neoplastic transformation.

Ascorbic acid inhibits replication and infectivity of avian RNA tumor virus.

Ascorbic acid, at nontoxic concentrations, causes a substantial reduction in the ability of avian tumor viruses to replicate in both primary avian tendon cells and chicken embryo fibroblasts. The virus-infected cultures appear to be less transformed in the presence of ascorbic acid by the criteria of morphology, reduced glucose uptake, and increased collagen synthesis. The vitamin does not act by altering the susceptibility of the cells to initial infection and transformation, but instead appears to interfere with the spread of infection through a reduction in virus replication and virus infectivity. The effect is reversible and requires the continuous presence of the vitamin in the culture medium.

Fujinami sarcoma virus: an avian RNA tumor virus with a unique transforming gene.

The oncogenic properties and RNA of the Fujinami avian sarcoma virus (FSV) and the protein it encodes were investigated and compared to those of other avian tumor viruses with sarcomagenic properties such as Rous sarcoma virus and the acute leukemia viruses MC29 and erythroblastosis virus. Cloned stocks of FSV caused sarcomas in all chickens inoculated and were found to contain a 4.5-kilobase (kb) and an 8.5-kb RNA species. The 4.5-kb RNA was identified as the genome of defective FSV because it was absent from nondefective FSV-associated helper virus and because the titer of focus-forming units increased with the ratio of 4.5-kb to 8.5-kb RNA in virus preparations. This is, then, the smallest known tumor virus RNA with a transforming function. Comparisons with other viral RNAs, based on oligonucleotide mapping and molecular hybridization, indicated that 4.5-kb FSV RNA contains a 5' gag gene-related sequence of 1 kb, an internal specific sequence of about 3 kb that is unrelated to Rous sarcoma virus, MC29, and erythroblastosis virus, and a 3'-terminal sequence of about 0.5 kb related to the conserved C region of avian tumor viruses. The lack of some or all nucleotide sequences of the essential virion genes, gag, pol, and env, and the isolation of FSV-transformed nonproducer cell clones indicated that FSV is replication defective. A 140,000-dalton, gag-related non-structural protein was found in FSV-transformed producer and nonproducer cells and was translated in vitro from full-length FSV RNA. This protein is expected to have a transforming function both because its intracellular concentration showed a positive correlation with the percentage of transformed cells in a culture and because FSV is unlikely to code for major additional proteins since the genetic complexities of FSV RNA and the FSV protein are almost the same. It is concluded that the transforming onc gene of FSV is distinct from that of Rous sarcoma virus and other avian tumor viruses with sarcomagenic properties. Hence, multiple mechanisms exist for sarcomagenic transformation of avian cells.

Phorbol ester-mediated stimulation of the synthesis of mouse mammary tumour virus

12-O-tetradeconoyl-phorbol-13-acetate (TPA) is a potent tumour promoter in the two-stage mouse skin carcinogenesis model1,2. Recently, TPA has been shown to enhance the transformation of cultured cells by two DNA tumour viruses—adenovirus3 and Epstein–Barr virus4. It is not known whether TPA also augments the transformation of cells by RNA tumour viruses. In Rous sarcoma virus (type C) transformed fibroblasts, the levels of plasminogen activator are elevated by TPA5,6, a phenomenon also associated with malignant transformation7,8. In addition, TPA and other promoters induce the synthesis of some herpesviruses in persistently infected cells9,10. I have investigated the effect of TPA on the synthesis of mouse mammary tumour virus (MMTV), type B RNA tumour virus, and find that TPA, in nanogramme quantities, causes a 10- to 20-fold stimulation of MMTV production by persistently infected mouse mammary tumour cells. This stimulation is specific for TPA; the non-promoter, phorbol, does not stimulate MMTV production.

Evidence for type-C retrovirus production by Burkitt's lymphoma-derived cell line.

Burkitt's lymphoma cell line, P3HR-I, was found to secrete virions with properties of known type-C RNA tumor viruses. The viral particles had a buoyant density of 1.16 g/ml in sucrose gradients and contained a high-molecular-weight RNA and an RNA-instructed DNA polymerase. The viral polymerase was active in an endogenous reaction requiring the presence of the four deoxyriboside triphosphates and manganese ions, and was sensitive to RNase. The DNA product of the endogenous reaction specifically hybridized to P3HR-I viral 60 to 70S RNA. Electron microscopic examination of ultrathin sections of P3HR-I cells revealed immature, mature and budding virions typical of type-C retroviridae. Nucleic acid hybridization assays showed no sequence homoblastosis virus, murine oncornaviruses, simian sarcoma virus or RD114 virus.

Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors.

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.

Avian RNA Tumour Viruses

Avian RNA Tumour Viruses

Efficient production of mammalian RNA tumor viruses in serum-free culture medium allows rapid RNA subunit purification

A wide variety of infected mammalian cell cultures were observed to produce high levels of RNA tumor virus particles in the absence of serum for at least 12 h. Virus production was measured by yields of 50–70 S viral RNAs isolated directly from serum-free culture media by chromatography on oligo(dT)-cellulose. Yields of RNAs from viruses produced in serum-free medium were comparable to yields obtained from purified viruses produced in serum-containing medium. Subunits of viral RNAs were thermally dissociated and separated by a new sedimentation system using sucrose gradients with resolving power (in the relevant size range) equivalent to that obtained with electrophoresis in polyacrylamide gels. RNA subunits isolated directly from serum-free medium appeared to be intact as judged by poly(A) content and resedimentation. The overall approach developed here represents dramatic savings in time and effort over previous ways of producing purified RNA subunits from RNA tumor viruses.

Presence of RNA methylases in intracisternal A particles purified from a mouse plasma cell tumor.

Intracisternal A particles share several biochemical properties with RNA tumor viruses, including 70‐S RNA, reverse transcriptase, and a group‐specific protein. In addition they form by budding from cytoplasmic membranes. It has been shown previously that various purified RNA tumor viruses contain N2‐guanine RNA methylase; purified A particles were tested to determine if they contained this activity as well. The results showed that instead of a single methylase, six methylases were associated with intracisternal A particles: a cytidine and a uridine methylase, two N2‐guanine monomethylases, an N2,N2‐dimethylguanine methylase and an adenine methylase. These enzymes were indistinguishable from the cellular enzymes when Escherichia coli tRNAPhe was used as an acceptor RNA and three were shown to have the identical specificity, i.e. N2‐guanine methylases for positions 10 and 34 and methylation of adenine in position 73 of the tRNAphe. The specific activity of the core‐associated RNA methylases increased approximately twofold over the methylases of the intact particles, indicating that these methylases were associated with the internal structure and not simply adherent to the envelope surface. This suggests that these cellular enzymes are packaged within intracisternal A particles during synthesis. Thus, intracisternal A particles are similar to the oncornaviruses in that reverse transcriptase is the only known enzyme not of cellular origin, but they appear to contain cellular methylases not found in other RNA tumor viruses.

Progenitor-cell populations can be infected by RNA tumor viruses, but transformation is dependent on the expression of specific differentiated functions.

Progenitor-cell populations can be infected by RNA tumor viruses, but transformation is dependent on the expression of specific differentiated functions.

8] Repair of overlapping DNA termini

This chapter deals with repair of overlapping DNA termini. The natural cohesive termini of bacteriophage DNA, such as γ and the staggered phosphodiester bond cleavages produced by various restriction endonucleases have several possible arrangements. If the arrangement is such that a protruding 5' single-stranded end and an internal 3'-hydroxyl end are formed, then the termini will serve both as primer and template for repair synthesis by a DNA polymerase. Overlapping DNA termini can be repaired using the RNA directed DNA polymerase, which is called reverse transcriptase, from RNA tumor viruses and radioactive nucleoside triphosphates. The incorporation into the termini can be quantified and the sequence analyzed from nearest neighbor data. The chapter further explains the labeling procedure.

Immunoselection of mutants deficient in cell surface glycoproteins encoded by murine erythroleukemia viruses.

We have described a heterogeneously processed glycoprotein with an apparent molecular weight of 55,000 (gp55) that is encoded by the Friend spleen focus-forming virus, an acute erythroleukemia virus [Dresler, S., Ruta, M., Murray, M.J. & Kabat, D. (1976) J. Virol. 30, 564-573]. Several lines of evidence suggest that a small proportion of the gp55 in infected cells is located on the surface membranes. First, different nonproducer cell lines infected with cloned Friend spleen focus-forming virus were efficiently killed in the presence of complement by cytotoxic antisera that react with gp55. Furthermore, a clone of cells selected for resistance to the cytotoxic antibody synthesized an altered intracellular form of gp55. This immunoselection procedure appears to also be useful for isolating glycoprotein mutants of other RNA tumor viruses. Analysis of cell surface proteins labeled with [125I]iodine supported the idea that gp55 occurs on the plasma membranes. However, the cell surface gp55 had more highly processed oligosaccharides than the majority of the gp55, which occurs within the infected cells. In addition, we have found that leukemia cells from mice infected with the erythroleukemic Rauscher virus complex contain a membrane glycoprotein that appears similar to the gp55 encoded by Friend spleen focus-forming virus. The encoding of similar glycoproteins by independently isolated acute erythroleukemia viruses suggests that these glycoproteins may be important in leukemogenesis.

Tumor virus and human cancer.

A substantial amount of data suggests that both RNA and DNA tumor viruses are associated with human cancer. Correction of malignant transformation at the genotypic level may be possible in the future but will require significant advances in our understanding of genomic material. The use of simple defined models such as tumor viruses seems like a reasonable place to start. The use of antipromotors (e.g. retinoids) and inhibitors of promotors will prove as important to the successful treatment of cancer in the next decade as chemotherapy has been in the last two decades.

Antibody response to Moloney type C virus-induced tumours.

Animals infected with RNA tumour viruses show an immune response to new virus-induced antigens present on the tumour cell surface. In tumours induced by murine leukaemia virus (MuLV), serologically detected cell membrane antigens have been placed into two major groups; the Gross cell-surface antigen(s), GCSA and GIX, are associated with Gross leukaemia virus infection1 and the FMR antigen (s), associated with infection by Friend, Moloney or Rauscher type C viruses2. There is good evidence that some of these antigens are related to the structural proteins of the virus. The GIX antigen is found on the viral envelope protein, gp70 (refs 3–5) and the GCSA antigen is on the p30 protein which is present in the membrane as part of the glycosylated ‘gag’ polyprotein(s)6–8. It is not certain whether the FMR antigen(s) is found on membrane-located viral structural precursors or polyproteins, or whether it is present on a tumour-associated protein. Several antigens of this latter type have been described. For example, the Moloney leukaemia virus-induced cell-surface antigen (MCSA) is thought to be a non-viral protein associated with ‘gag’ products, p30, p15 and p12 on the membranes of Moloney lymphoma cells9,10. MCSA and the antigens on cells infected by Abelson MuLV11 may be very specific cellular antigens associated with Moloney MuLV infection. There has also been much interest in whether the src gene product is present in the membrane of sarcoma virus-induced tumours, although it is thought not to be present on the external membrane surface12. A more controversial point is whether there are other virus-induced cell-surface antigens (CSA) which may serve as targets for an effective immune response to viral tumours13. We have previously found14 that serum from BALB/c mice with regressed Moloney virus-induced fibrosarcomas contained specificities only for Moloney viral proteins and not for any tumour-associated protein like MCSA. We have now identified the viral proteins with which the Moloney MuSV antisera react. Using a syngeneic system of immunisation with freshly induced Moloney sarcoma tumours, we show that the antibody response is directed only at viral structural antigens and that the major part of the response is to antigens on p30 protein probably present as part of a precursor polyprotein on the tumour cell surface.