Separation of sarcoma virus-specific and leukemia virus-specific genetic sequences of Moloney sarcoma virus.

We have studied the nucleic acid sequences in nonproducer cells transformed by Moloney sarcoma virus or Abelson leukemia virus (two types of replication-defective, RNA-containing, viruses isolated by passage of Moloney leukemia virus in BALB/c mice). DNA probes from the Moloney leukemia in virus detect RNA in both Abelson virus-transformed nonproducer cells and Moloney sarcoma virus-transformed nonproducer cells. A sarcoma-specific cDNA, prepared from the Moloney sarcoma virus, has extensive homology to RNA found in heterologous nonproducer cells transformed by Moloney sarcoma virus, has little homology to RNA in cells producing Moloney leukemia virus, and no detectable homology to RNA in nonproducer cells transformed by the Abelson virus. By analogy to earlier data on avian and mammalian sarcoma viruses, these results suggest that the Moloney sarcoma virus arose by recombination between a portion of the Moloney leukemia virus genome and additional sarcoma-specific information, and indicate that the expression of this information in not essential for Abelson virus-mediated fibroblast transformation.     

Demonstration of a Cell-Surface Antigen Associated with Murine Sarcoma Virus by Immunoelectron Microscopy

Cells transformed by murine sarcoma virus have been examined for the presence of a new virus-associated cell-surface antigen by immunoelectron microscopy. A common antigen has been detected on the surface of nonproductively transformed cells that were induced by two different strains of murine sarcoma virus, Kirsten and Moloney. This antigen shows crossreaction with cell lines transformed by murine sarcoma virus that were produced in two different mammalian species, rats and mice. Further, this antigen is distinct from previously described antigens on the surfaces of cells infected by murine leukemia virus, on the viral envelope, and on the surfaces of spontaneously transformed cell lines or cell lines transformed by x-irradiation.     

Control of reversion of Moloney virus sarcoma virus transformed cells.

Mouse cells transformed by the Moloney murine sarcoma virus (MSV) native to the species can give rise to revertants which are supersusceptible to a second cycle of transformation. The MSV retransformed cells can give rise to several complex functional states even after several cycles of cloning: a) Formation of sarcoma positive, leukemia negative (S+Lminus) type cells of which some sublines may be inducible for MSV by halogenated pyrimidines; b) detection of initially SminusLminus cells which spontaneously become transformed and S+Lminus; c) the derivation of flat murine leukemia virus positive (MuLV+) cells which have an atypical MuLV and may become MSV+ as well as MuLV+; d) the release of free MSV from some cell clones with an apparent absence of MuLV. A general feature of all the above variants is a failure of detection of spontaneous reversion occurring after the second cycle of transformation. The nature of MuLV spontaneously released is that of a poorly replicating MuLV which exhibits cross-interference with other MuLV pseudotypes of MSV and the envelope which is most similar to that of Moloney leukemia virus (MLV). The examination for spontaneous reversion in human S+Lminus cells transformed by the same type of genome capable of good frequency of reversion in mouse cells, did not yield human revertant cells.     

Temperature-Dependent Expression of Transformation by a Cold-Sensitive Mutant of Murine Sarcoma Virus

Morphological transformation of normal rat kidney cells by a murine sarcoma virus was found to be cold-sensitive. Cells transformed by the virus expressed their transformed phenotype at the permissive temperature (39 degrees ) but not at the nonpermissive temperature (33 degrees ), as judged by the criteria of morphological changes and colony-forming ability on monolayers of normal rat kidney cells. Cold-sensitive expression of transformation was specific for focus-derived normal rat kidney cells transformed by the virus, readily reversible, and not lost during serial propagation of the cells. The genome of the murine sarcoma virus can be rescued by superinfection with Moloney leukemia virus at the permissive or nonpermissive temperature, and the rescued virus exhibited the same cold-sensitive properties as the original transforming virus. These results suggest that maintenance of the transformed state is continuously dependent on a cold-sensitive viral function.     

Tissue tropism of a leukemogenic murine retrovirus is determined by sequences outside of the long terminal repeats.

Although it has been previously determined that the long terminal repeat (LTR) sequences of several murine retroviruses specify the major tissue tropism of leukemias they induce, data reported here show that the LTR is not responsible for tissue tropism in the case of all leukemogenic viruses. In an effort to determine whether LTR sequences of the acute erythroleukemia-inducing spleen focus-forming virus (SFFV), like those of the other murine leukemia viruses, are uniquely required to confer tissue specificity to the virus, we prepared recombinant SFFVs in which the LTR region containing promoter and enhancer functions was replaced with analogous LTR regions from Friend and Moloney ecotropic and mink cell focus-inducing viruses. It was found that all of the SFFV constructs, even those with a LTR derived from lymphoma-inducing viruses such as Moloney murine leukemia virus, transformed erythroid cells in vitro and induced exclusively an erythroid disease. These results demonstrate that sequences in SFFV that determine the tissue-specific nature of the disease reside outside the LTR.     

Control of growth by picolinic acid: Differential response of normal and transformed cells

Picolinic acid reversibly inhibits the growth of cultured cells. Fourteen other pyridine derivatives were ineffective or toxic. Untransformed normal rat kidney (NRK) cells are reversibly arrested in the G(1) stage of the growth cycle as shown by cell counts, mitotic index, [(3)H]thymidine incorporation, and flow microfluorometry. Flow microfluorometry was used to monitor the effects of picolinic acid on numerous other cell lines. Normal cells are blocked in G(1), whereas transformed cells show responses that are dependent upon the transforming virus and independent of species or origin of the cell line. Kirsten sarcoma virus-transformed cells are blocked in G(1). Simian virus 40-transformed cells progress to a G(2) block. Cells transformed by polyoma or Harvey sarcoma virus with Moloney virus coat have flow microfluorometry profiles that indicate blocks in both G(1) and G(2). Cells transformed with Moloney sarcoma virus are not blocked in a specific phase of the cell cycle. Picolinic acid does not change the levels of NAD(+) plus NADH; however, the growth inhibition by picolinic acid is partially overcome by nicotinamide. These results suggest that picolinic acid interacts with a specific growth control mechanism that may involve NAD(+) and that this control mechanism is altered by different transforming viruses in different manners.     

Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse.

Antisera prepared against BALB/c Meth A sarcoma in syngeneic or compatible F1 mice recognize a protein with an apparent molecular weight of 53,000 in extracts of [35S]methionine-labeled transformed BALB/c cells. This component, designated p53, was not detected in normal adult mouse fibroblasts, lymphoid cells, or hematopoietic cells or in mouse embryo cells or 3T3 cells. An extensive variety of antisera, including alloantisera and heterologous antisera directed against structural antigens of murine leukemia viruses, was tested for reactivity with p53; other than Meth A antisera, only comparably prepared antisera against another BALB/c sarcoma, CMS4, had anti-p53 activity. All transformed mouse cells tested were found to express p53; these tests included chemically induced sarcomas, leukemias, spontaneously transformed fibroblasts, and cells transformed by simian virus 40 and murine sarcoma virus. The presence of p53 in tumors of no known viral etiology indicates coding by resident cellular genes; this does not exclude endogenous viruses as the source of coding sequences or the possibility that transforming viruses code directly for p53.     

MURINE SARCOMA VIRUS TRANSFORMATION OF BALB/3T3 CELLS: LACK OF DEPENDENCE ON MURINE LEUKEMIA VIRUS

Murine sarcoma virus induces foci of morphologically altered cells in BALB/3T3 cultures. Focus formation in mouse cells has been thought to require the presence of a helper, murine leukemia virus, which is present in murine sarcoma virus stocks but by itself does not induce any morphological transformation of mouse cells. The present studies show that early after infection, the titration pattern for murine sarcoma virus in BALB/3T3 cells is "two-hit" since only foci produced by virus spread can be detected. Such foci require the presence of both viruses in the initially infected cell. By seven days the titration pattern is "one-hit" under culture conditions which allow the growth and detection of small foci of transformed cells induced by murine sarcoma virus alone. The "two-hit" titration pattern results from the inability to detect these foci. We conclude that murine sarcoma virus is able to transform mouse cells without requiring murine leukemia virus.     

Inhibition of Murine Leukemia Virus Replication by Poly(vinyluracil) and Poly(vinyladenine)

Poly(1-vinyluracil) and poly(9-vinyladenine), as well as the corresponding polynucleotides poly(uridylate) and poly(adenylate), inhibit acute murine leukemia virus infection in mouse-embryo cells, but they do not significantly inhibit the replication of Sindbis and vesicular stomatitis viruses. The polymers were most effective as inhibitors when added during an early stage of virus replication. Effects of vinyl polymers on the RNA-dependent DNA polymerase from the virions of murine leukemia virus were also observed.     

Effects of Poly(2′-O-Methyladenylic Acid) on Susceptibility and Autogenous Immunity to RNA Tumor Virus Oncogenesis In Vivo

Poly(2'-O-methyladenylic acid) [poly(Am)] inhibited tumor development and death induced by the Moloney sarcoma virus-leukemia virus complex in newborn mice. The compound was effective at 10 mug per mouse when given at least 1 hr before inoculation of virus, but the greatest inhibition was seen in mice treated at least 4 hr before infection. Poly(2'-O-methyluridylic acid) and poly(vinyladenine) also inhibited sarcoma development and death but were less effective than poly(Am). Poly(Am) also enhanced the antibody response of newborn mice to endogenous leukemia virus envelope antigens, which we refer to as autogenous immunity. The results of these preliminary studies suggest that poly(Am) altered the oncogenic potential of the Moloney sarcoma-leukemia virus complex in vivo, and the effect appears to be mediated through an enhancement of the immune response of the treated animals.     

VIRUS-INDUCED SARCOMA OF MICE: INHIBITION BY A SYNTHETIC POLYRIBONUCLEOTIDE COMPLEX

The availability of a potent inducer of interferon, the synthetic double-stranded RNA (poly I.poly C), prompted us to determine its possible prophylactic and/or therapeutic effect on virus-induced sarcomas of mice. When treatment was begun prior to virus inoculation and repeated on alternate days, the majority of NIH Swiss mice inoculated with Moloney or Friend pseudotypes of Moloney murine sarcoma virus failed to develop tumors. Other experiments suggested that repeated injections initiated even after the establishment of tumor nodules were also effective. Mice injected on the day of birth with poly I.poly C develop high titers of interferon. The evidence favors, but does not establish, the interpretation that the observed tumor inhibition is mediated through the induction of endogenous interferon. These experiments demonstrate that treatment with poly I.poly C is followed by a regression of established murine sarcoma infection in mice.