Murine RNA Tumor Viruses Research Articles

Carcinomas induced by cell lines cultivated from normal mouse placentas.

Cell lines have been established from placentas of various strains of mice by in vitro cultivation. The established lines appear to be trophoblast cells as judged from their production of gonadotropin-like substance and their production of gonadotropin-like substance and steroid hormones. The cell lines lack detectable H-2 antigen, Fc receptor sites, Thy 1 and antigen and surface immunoglobulin determinants. In addition, the cells are resistant to murine type-C RNA tumor virus infection. The resistance is due to a block of viral replication after the stages of viral adsorption and penetration. The cell lines induced carcinomas after injection into adult mice of original host strains. Usually at least 2 x 10(6) cells are required to induce tumors by i.p. or s.c. injection. Transplantation of the tumor cells to various strains of mice revealed that some strains accepted and some strains rejected the transplant. Genetic crossings between susceptible and resistant mouse strains indicate that susceptibility to transplantation of tumor is determined by a single pair of dominant autosomal genes.

54] Assay of effect of interferon on viruses that bud from plasma membrane

This chapter studies the effect of low doses of IF (3-30 units/ml) on vesicular stomatitis virus (VSV) infection of L cells. It is observed that a marked inhibition in glycoprotein (G) and membrane protein (M) of VSV released from IF-treated cells. Evidence supporting the selective deficiency of glycoprotein in VSV released from IF-treated cells is also derived from electron microscopy studies. These results suggested that VSV-particles released from IF-treated cells are low in infectivity. The results in many respects resemble those that were previously reported in IF-treated cells infected with murine leukemia viruses (MLV). It is concluded that such findings on the action of interferon are not limited to murine RNA tumor virus systems; and perhaps the inhibition of some membrane associated viruses (such as VSV) by treatment with relatively moderate concentrations of IF may be widespread phenomenon that is closely related to functional abnormalities in the proteins incorporated into noninfectious virions produced by such cells. The effect of IF on membrane-associated virus, are presented. The classes listed may involve successively less severe degrees of alteration in viral proteins; these alterations could result in inhibition of virus assembly or infectivity at later stages in the replication cycle.

Serological analysis of cellular and viral DNA polymerases by an antiserum to DNA polymerase gamma of human lymphoblasts.

An antiserum has been prepared against a highly purified DNA polymerase gamma from NC37 cells, a normal human lymphoblast cell line. The antiserum does not possess enzyme neutralizing activity, but does bind specifically to DNA polymerase gamma. When tested in a double antibody immunoprecipitation assay, the antibody does not cross-react with DNA polymerases alpha or beta, purified from NC37 cells, or with reverse transcriptases of avian, murine, or primate RNA tumor viruses. Antisera prepared against purified reverse transcriptases similarly do not recognize DNA polymerase gamma, either in an enzyme neutralization assay or in the more sensitive double antibody immunoprecipitation assay. The availability of an antiserum to DNA polymerase gamma will allow the further characterization of enzyme activities isolated from cellular material and suspected of being related to viral reverse ttranscriptases. In those cases where such activities do not immunologically resemble known viral DNA polymerases, the anti-DNA polymerase gamma will help determine the viral or cellular nature of the unknown activity.

Inactivation of murine RNA tumor viruses by isatin beta-thiosemicarbazone, its derivatives and analogs.

Abstract Isatin beta-thiosemicarbazones inactivate the transforming ability of murine sarcoma virus and the replication of murine leukemia virus (MuLV) upon direct contact in vitro. The drugs inhibit the RNA-dependent DNA polymerase of MuLV. Cell transformation of MSV is also inhibited when infected cells are exposed to certain concentrations of the drug. Leukemia in mice was enhanced in drug-treated mice. This effect is probably due to inhibition of the immune response by the drug.

A comparison of the enzymatic responses of the DNA polymerases from four RNA tumor viruses

DNA polymerases from avian, feline, murine and simian RNA tumor viruses exhibit substantial differences in optimal assay conditions and vary widely in their template-primer preferences. Avian DNA polymerase utilizes both natural and synthetic template-primers efficiently in the presence of Mg ++ as well as Mn ++. By contrast, the mammalian viral DNA polymerases are much more responsive to poly(A)·oligo(dT) than to other template-primers, and exhibit up to 20-fold greater activity with Mn ++ than with Mg ++. In addition, simian sarcoma virus DNA polymerase shows no detectable response to poly(C)·oligo(dG) over a wide variety of conditions stimulatory to the other viral enzymes.

Synthesis of long, representative DNA copies of the murine RNA tumor virus genome.

Virions of Moloney murine leukemia virus can synthesize two classes of DNA molecules complementary to their 70S RNA. One class consists of molecules about 200 nucleotides long, which are of limited sequence complexity; these molecules are formed preferentially if the dNTP concentration during the reaction is low. The second class consists of very heterogeneous DNA molecules with weight-average size of about 1,000 nucleotides containing at least 70% of the viral RNA sequences in approximately equal concentration. The longest of these molecules can be 5,000 nucleotides long. This second class of DNA is formed in large amounts only in reactions containing dNTP concentrations of 0.2 mM or higher. In such reactions after 24 h of incubation, at least 35% of the input RNA is represented in DNA copies. The ability to make long, representative DNA transcripts of tumor virus RNA provides a source of excellent probes for molecular hybridization.

Isolation of type C virions from a normal human fibroblast strain.

Type C virions were spontaneously released from cultures of a diploid human cell strain. The varions have properties of known type C RNA tumor viruses and share antigenic determinants with the major interspecies-specific antigen (p30) of simian sarcoma virus. Antiserum to reverse transcriptase of gibbon ape leukemia virus inhibits the reverse transcriptase of the putative human virions and that of simian sarcoma virus, but has no effect on the corresponding enzymes of avian or murine RNA tumor viruses.

Detection of reverse transcriptase in human breast tumours with poly(Cm)-oligo(dG).

PARTICLES having the density of type-C RNA tumour viruses, possessing reverse transcriptase (RNA-directed DNA polymerase) activity, and containing high molecular weight RNA that shares a portion of its base sequences with the RNA of murine RNA tumour viruses, have been reported to be present in several types of human malignant tissues, but not in their normal counterparts (review, see ref. 1). The endogenous RNA→DNA synthetic capabilities of these particles were used in a simultaneous detection test2,3 to determine these parameters. Because of their similarity and relatedness to animal oncornaviruses, these particles are considered to be possible aetiological agents of human cancer2.

Lack of requirement of reverse transcriptase function for the activation of murine leukemia virus by halogenated pyrimidines

Polynucleotides, poly(adenylate) and poly(uridylate), and corresponding vinyl polymers, poly(vinyladenine) and poly(vinyluracil), which were shown to inhibit in vitro murine RNA tumor virus reverse transcriptase, do not effect the induction of AKR virus by IdUrd from virus negative AKR cells. Under identical conditions, exogenous infection by AKR virus in these cells was markedly inhibited. These results suggest that the function of virus specific reverse transcriptase is not essential for the induction process.

Zinc reverse transcriptases from mammalian RNA type C viruses

1,10-phenanthroline (OP) reversibly inhibits the RNA dependent DNA polymerases reverse transcriptases from murine, simian, feline and RD-114 RNA tumor viruses. The log of the OP inhibition constants, pKI, vary from 4.7 to 4.9 and the number of moles of inhibitor bound to the metal, ii, vary from 1.3 to 2.2. These values are very similar to those previously reported for known zinc enzymes and imply that the mammalian reverse transcriptases are also zinc enzymes. Combined use of microwave-induced emission spectrometry and micro gel exclusion chromatography demonstrates the presence of stoichiometric amounts of zinc in the enzymes from murine leukemia and woolly monkey type C-viruses. These results parallel those previously reported for the enzyme from avian myeloblastosis virus.

Pseudotype formation between enveloped RNA and DNA viruses.

ENVELOPED RNA viruses from different groups participate readily with each other in phenotypic mixing during maturation at the plasma membrane of host cells. These ‘pseudotypes’ with the envelope antigens of one virus and the genome of the other have been demonstrated for simian virus 5 and vesicular stomatitis virus (VSV)1, for avian or murine RNA tumour viruses and VSV2–4, and for fowl plague virus and VSV5. (An exception has been the lack of interaction between Sindbis virus and VSV6.) We have now found virions containing the RNA genomes of VSV in the envelope antigens of a DNA virus, herpes simplex virus (HSV). These interactions between genomes and antigens of unrelated groups may be part of a mechanism for viral pathogenesis and oncogenesis.

Mechanism of Stimulation of Murine Type-C RNA Tumor Virus Production by Glucocorticoids: Post-Transcriptional Effects

We have previously shown that dexamethasone stimulates production of type-C virus from seemingly normal murine fibroblasts (BALB/3T3) and from transformed (Kirsten sarcoma-leukemia virus) nonproducing cells (BALB/K3T3) induced by 5-iododeoxyuridine. In this report, we further examine the mechanism of this effect by using BALB/K3T3 cells. Several observations suggest that this effect is post-transcriptional. The optimal stimulation by dexamethasone is obtained when dexamethasone is given 24 to 48 h after 5-iododeoxyuridine induction. Although this effect is late, time course experiments suggest that dexamethasone does not act to promote release of preformed virions. The stimulation by dexamethasone is blocked when cells are treated with cordycepin (3'-deoxyadenosine) during the first 24 h of induction, but not when cordycepin is added later. Conversely, interferon, which inhibits virus production, interferes with dexamethasone when it is added late or after removal of the steroid. The results of molecular hybridization experiments show that there is no detectable increase in Kirsten sarcoma-leukemia virus-specific RNA in dexamethasone-treated cells (with or without 5-iododeoxyuridine). The results of the time course studies, and the cordycepin, interferon, and hybridization experiments, suggest that the effect of dexamethasone on type-C virus production in this system is post-transcriptional.

Inhibition of murine RNA tumor virus replication and oncogenesis by chloroquine.

AbstractThe effects of 7‐chloro‐4‐(4‐diethylamino‐l‐methylbutylamino) quinoline diphos‐phate (chloroquine) on marine RNA oncogenic virus infection and replication were studied in cell culture and in vivo. Replication ofMoloney leukemia virus in cell culture was inhibited by approximately 75% at a drug concentration of 1 s̈/ml. This concentration had no effect on cellular DNA or RNA synthesis or on cell division. Chloroquine at a dose of 50 mg/kg body weight given 15 min after injection of Moloney murine sarcoma virus prevented tumor development in newborn mice; similar treatment inhibited the induction of splenomegaly by Rauscher leukemia virus in adult mice. The effect of the drug is not exerted on the virus particle itself, since incubation of the virus with chloroquine for 30 min prior to infection did not decrease its oncogenicity. Presumably, the drug affects one or more events in virus replication.

Systematics of RNA tumor viruses and virus-like particles of human origin.

[(3)H]DNA copies of avian, feline, murine, and primate RNA tumor virus genomes were synthesized in vitro by an RNA-dependent DNA polymerase reaction. These DNAs were hybridized to 60-70S RNA that had been purified from the viruses. The amount of the [(3)H]DNA hybridized yielded a measure of the genetic relatedness among the DNA preparations synthesized by the viruses. When many combinations of DNA and RNA were analyzed, the pattern of hybridization showed in some cases that the DNA copies of the viral RNA were related to each other in the same way that the natural hosts of the viruses are phylogenetically related. This pattern was observed only among the RNA leukemia viruses. The sarcoma component in sarcoma-leukemia viruses from rats and primates appeared to be unusually closely related. The mouse mammary carcinoma virus and two unclassified viruses (MPMV and Visna) appeared to be genetically distinct.A similar analysis of DNA synthesized by an RNA-dependent DNA polymerase associated with a viral-like particle obtained from the cytoplasm of human leukemic white blood cells demonstrated that this DNA occupied a space in the affinity pattern of leukemia viruses which is expected of a nucleic acid from a primate-type-C RNA tumor virus. This observation strengthens earlier evidence that components of RNA tumor viruses are associated with human leukemia.

Separation of ribonuclease H and RNA directed DNA polymerase (reverse transcriptase) of murine type-C RNA tumor viruses.

Ribonuclease H (RNA.DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) has been reported to copurify with reverse transcriptase (RNA directed DNA polymerase) of RNA tumor viruses. In addition, viral specific ribonuclease H and reverse transcriptase of avian type-C viruses are thought to be part of the same polypeptide. In this report we show that a fraction of the ribonuclease H activity from Rauscher murine leukemia and Kirsten murine sarcoma viruses was separated from reverse transcriptase by anion exchange chromatography while the remaining portion co-purified with the viral polymerase. The amount of this co-purified nuclease activity was about 4- to 8-fold lower than the activity found in avian myeloblastosis virus (with respect to the ratio of ribonuclease H to reverse transcriptase) and this nuclease activity can only be detected by using labeled substrate of high specific radioactivity. However, a complete separation of ribonuclease H activity from reverse transcriptase was obtained by purifying core structures of the virus by sucrose density gradient centrifugation. While reverse transcriptase was present in the cores, there was no detectable ribonuclease H. Furthermore, a specific antibody against Rauscher leukemia virus reverse transcriptase did not inhibit any virion associated ribonuclease H activity. Our results suggest that in these virions these two enzyme activities reside in two separate molecules and probably in two different compartments of the virus. These findings emphasize a basic difference between the avian and murine type-C virus DNA polymerases.

RNA-dependent DNA polymerase associated with a simian lymphoid cell line derived from a Herpesvirus saimiri-induced lymphoma.

AbstractThis communication describes the spontaneous appearance of a membrane‐bound RNA‐dependent DNA polymerase in a simian lymphoma cell line that was established from a lymphoma tumor mass previously induced by Herpesvirus saimiri in an owl monkey. This membrane‐bound RNA‐dependent DNA polymerase fraction possesses many biochemical properties analogous to those of known primate and murine RNA tumor viruses. Characteristics of this RNA‐dependent DNA polymerase are clearly distinct from those of the DNA polymerase of Herpesvirus saimiri. This particular fraction incorporates tritiated ribonucleosides into RNA in an in vitro system, and it sediments at 1.15–1.16 g/cm3. It is not induced to greater production by halogenated deoxyuridines. The current observations were made at a time when the host cells were no longer producing Herpesvirus saimiri.

Remarks on the Ultrastructure of Type A, B, and C Virus Particles

Publisher Summary The scope of this chapter is limited to the recent findings concerning the ultrastructure of murine RNA tumor viruses, as they appear after various preparatory procedures under the electron microscope and to some implications of these observations in the continuing search for viruses of this type in human malignancies. Here attention is focused mainly on the literature that has appeared since 1968. The title of this chapter is in direct reference to the ultrastructural classification of the murine tumor viruses originally proposed by Bernhard and Guerin in 1958. The original classification contained some ambiguity in regard to the presence or absence of viral envelopes, Type A particles were originally described as having an electron-lucent center (the “doughnut-shaped” nucleoid), and as being located either within the cytoplasm (“intracellulaire”) or within the extracellular spaces (“inter-cellulaire”). When the budding process of these viruses was described, it became clear that the cytoplasmic and extracellular type A particles differ mainly by the acquisition of an envelope derived from the plasmalemma of the infected cell. Therefore, calling type A particles before or after budding by the same name would amount to ignoring an important step in the final assembly of the virus. To distinguish between the type A particles with or without an envelope, and at the same time to respect the original terminology of Bernhard and Guerin, it was later proposed to call the first “enveloped” A particles and the second “naked” A particles. It appears therefore that, in view of the uncertainties that persist on the identification of the RNA tumor viruses in human neoplasia, the results obtained by electron microscopy, which can often provide undisputed evidence for the presence of such agents, should be reviewed and the values of different preparatory procedures reevaluated.

Effects of Polyadenylic Acids on Functions of Murine RNA Tumor Viruses

Single-stranded polyribonucleotides, which competitively inhibit murine RNA tumor virus reverse transcriptase in vitro, were tested as inhibitors of various virus functions in cell culture. The compounds had two concentration-dependent effects. At high concentrations (100 mug/ml), both poly(adenylic acid) [poly(A)] and poly(2'-O-methyladenylic acid) [poly(Am)] inhibited the uptake of radioactively labeled leukemia virus by Swiss mouse embryo cells, but neither had a similar effect on Sindbis virus adsorption. At low concentrations (10 mug/ml), poly(Am) did not inhibit the uptake of leukemia virus but did inhibit virus replication by 85%; in contrast, the replication of Sendai virus and Sindbis virus was not inhibited significantly at this concentration. Both compounds were effective only when added prior to or early during virus infection. Poly(Am) was a much more effective inhibitor than poly(A), probably due to the nuclease resistance of the former compound. Poly(Am) at 5 mug/ml also inhibited transformation of 3T3 cells by Moloney sarcoma virus. However, neither poly(A) nor poly(Am) at high concentrations inhibited the activation of endogenous leukemia virus by iododeoxyuridine in AKR mouse embryo cells. These results suggest that virus reverse transcriptase plays an essential role in both the replication of exogenous murine leukemia viruses and the transformation of cells by murine sarcoma viruses but probably has no role in the activation of endogenous leukemia virus.

Growth of pseudotypes of vesicular stomatitis virus with N-tropic murine leukemia virus coats in cells resistant to N-tropic viruses.

Formation of pseudotypes between murine RNA tumor viruses and vesicular stomatitis virus (VSV) has been confirmed. Pseudotypes of VSV genomes coated by the surface envelope from an N-tropic tumor virus grew equally well in cells homozygous for either the Fv-1(n) or Fv-1(b) alleles. Therefore, the product of the Fv-1 locus, which restricts growth of murine RNA tumor viruses, must act on an intracellular aspect of tumor virus replication, a step after attachment and penetration.

Structural characteristics of some murine RNA tumor viruses studied by lactoperoxidase iodination.

Iodination by the noninvasive enzymatic lactoperoxidase technique has been used to study the enzyme-accessible and enzyme-inaccessible proteins of three oncorna viruses (radiation leukemia virus, Moloney leukemia virus, and mouse mammary tumor virus). The number and relative molecular weight of proteins associated with virion preparations purified on sucrose gradients were characterized by scans of Coomassie blue-stained bands after dodecyl sulfate-polyacrylamide gel electrophoresis. Gel scans from the leukemia viruses are similar, each showing six distinct major protein bands on stained gels. The mammary tumor virus proteins by this analysis are not similar to those of the leukemia viruses. Enzymatic iodination of intact virion preparations led to the solitary labeling of one of the major proteins of each virus-the 80,000-dalton protein of the leukemia viruses and the 52,500-dalton protein of the mammary tumor virus. These are tentatively positioned as surface (enzyme-accessible) moieties in the virions. Disruption of each virus with a nonionic nonionic detergent before enzymatic iodination led to the labeling of the remaining stainable bands. The four lower molecular weight bands of the leukemia viruses are tentatively positioned as internal (enzyme-inaccessible) components.