Shigeko Nomura

Revertants of mouse cells transformed by murine sarcoma virus: I. Characterization of flat and transformed sublines without a rescuable murine sarcoma virus

Abstract Murine sarcoma virus (MSV)-transformed mouse clonal cell lines produced variants with some properties of nontransformed cells. Such variant cells were epithelioid, contact inhibited, and grew to low density, and their low cloning efficiency in soft agar was similar to that of normal parental 3T3 cells. However, they contained murine leukemia (MuLV) group-specific antigen(s) without demonstrable virus production and reverse transcriptase activity. MSV could no longer be rescued from these flat variant cells by superinfection with MuLV, by cocultivation with normal 3T3 cells or by transspecies rescue into cat cells. An enhancement of sensitivity to MSV and MuLV infection was observed in all flat variant cultures. Flat variant clones spontaneously gave rise to retransformed cells during extended cultivation. Morphology, saturation density, and cloning efficiency in soft agar of cloned spontaneous retransformed cell lines were similar to the original MSV-transformed cells. However, they failed to demonstrate MuLV gs antigen(s), virus production, reverse transcriptase activity and a rescuable MSV genome. The spontaneously retransformed cells were susceptible to MSV and MuLV infection. After treatment with 5-iododeoxyuridine (IUrd), reverse transcriptase activity and virus particles were only rarely induced in flat variant or spontaneously retransformed clones. These particles were not infectious for the original host cells and were not induced in normal 3T3 cells or a majority of the variant clones. Chromosome studies of these variants suggested that the partial or complete loss of expression of transformation in variants might have been associated with an imbalance in the number of chromosomes mediating expression or suppression in these cells.

Efficient release of murine xenotropic oncornavirus after murine leukemia virus infection of mouse cells

Abstract A simple and a most efficient method of eliciting murine xenotropic type oncornavirus from virus-free mouse cells is an infection of such cells with murine ecotropic oncornavirus. Xenotropic virus appeared after a single passage of Moloney leukemia virus not only from 3T3 type cells of inbred mice but also from 3T3 cells of outbred Swiss mice and from wild mouse SC-1 cells which were not inducible by halogenated pyrimidines. Thus unexpected contamination of mouse oncornaviruses with related viruses of a broader host range is possible.

Revertants of mouse cells transformed by murine sarcoma virus: II. Flat variants induced by fluorodeoxyuridine and colcemid

Abstract Mouse sarcoma virus-transformed 3T3FL cells (S+L− cells) produced spontaneously and at variable rates flat variants with some properties of nontransformed cells. The frequency of occurrence of such variant cells increased after treatment of S+L−cells with fluorodeoxyuridine (FdUrd) or Colcemid. In one S+L− subline (3–360), the spontaneous flat variants (S) occurred at the rate of about 1 in 80 clonal colonies, and, after treatment with Colcemid or FdUrd, flat variants were observed in 1 of 20 and 30 colonies, respectively. In another S+L− subline (3–321), S variants normally occurred less than 1 in 1000 colonies, and the same treatments increased the frequency to approximately 1 in 70 and 55 colonies, respectively. Both the FdUrd and Colcemid-induced flat variants (F and C) resembled 3T3FL cells morphologically, grew to low saturation densities, and exhibited cloning efficiencies in soft agar which were 10 −1 to 10 −4 times lower than that of S+L− cells. However, except for one S subline, they contained murine leukemia group-specific antigen (s) without demonstrable virus production and reverse transcriptase activity. Murine sarcoma virus (MSV) was no longer rescuable by superinfection with murine leukemia virus (MuLV) or by cell fusion with 3T3PL, BALB/3T3, or normal rat kidney cells. All flat variant cells were susceptible to MSV and MuLV infection, and some degree of enhancement of sensitivity to MSV and MuLV infection was observed in most variant cultures. All flat variant sublines possessed the property of agglutinability by concanavalin A as high as that of S+L− cells. Some flat variant clones spontaneously underwent retrans-formation during extended cultivation. In contrast to previously described S+L−cell revertants, which spontaneously retransformed morphologically without a rescuable MSV genome, in the present experiments retransformation with a rescuable MSV genome was observed in 1 of 4 S sublines and 2 of 5 F sublines, but in none of 4 C sublines. Furthermore, 1 of 5 F sublines gave rise to a clone which was flat but which otherwise resembled S+L− cells. In the S and C variants, the loss of expression of transformation was associated with an increase in chromosome number. However, the chromosome number of F variants was similar to or slightly less than that of parental S+L− cells. These studies demonstrated that the reversion of S+L− cells to flat variants occurred by different mechanisms apparently involving either the viral or cellular genes or both, and that some flat variant sublines had retained at least one complete MSV genome in nondetectable form.

Revertants of mouse cells transformed by murine sarcoma virus: III. Metastable expression of virus functions in revertants retransformed by murine sarcoma virus

3T3FL cells transformed by the Moloney murine sarcoma virus (MSV) (sarcoma positive, leukemia negative, S+L- cells) segregate spontaneous flat revertants. These S-L- revertants do not release MSV upon superinfection with MuLV but are susceptible to a second cycle of single-hit MSV transformation. The behavior and nature of second-cycle MSV transformed revertants were followed by successive clonal generations. The second transformation by MSV resulted in a number of metastable states. The simplest of these was a tranformed S+L- like state, which in contrast to parental S+L- cells, was also inducible by halogenated pyrimidines. A second type consisted of a transformed S+L- clone which could give rise to a secondary flat S-L- clone, which when followed over several generations could turn again into a stable S+L- type cell. Thus an MSV genome can persist in a nontransformed cell in a nonrescuable state. A third condition has been found in which a previously S+L- type MSV retransformed revertant began to produce infectious MSV with or without an atypical murine leukemia virus (MuLV). Other clonal sublines from a second cycle MSV transformed cell resulted in both a stable S+L- type line and a spontaneous MSV releaser line. The S+L- type retransformed cell resembled parental single cycle MSV transformed S+L- cells in that it contained MuLV group specific antigen(s), and C type particles. Some second cycle MSV transformed S+L- cells produced infectious MSV on chemical induction. No second-cycle reversion was ever observed, if both the first and the second infecting genomes were Moloney-MSV. The nature of the atypical MuLV was examined in terms of host range, growth potential, and antigenicity. This MuLV was at times released with an apparent excess of MSV, yet if eleven weekly blind passages of such a virus stock were made in S+L- cells, replicating MuLV was found in titers similar to the MSV content. This atypical virus replicated in mouse cells, less in rat cells, but not at all in human or cat cells. Neutralization revealed that it was closely related to the Moloney type of MuLV. The above observations are compatible with the interpretation that the second cycle of MSV transformation destabilizes cellular controls over the transforming genome.

Induction of a novel transforming virus (MSV-Z) from sarcoma-positive leukemia-negative 3T3FL mouse cells

Abstract The S+L− cell lines derived from Swiss mouse 3T3FL cells transformed by Moloney murine sarcoma virus (M-MSV) in the absence of mouse-tropic murine leukemia virus (MuLV) produce type-C viral particles which are not infectious for a variety of mammalian cells. A rescuable MSV genome is present in such cell lines. We report here that preparation of particles spontaneously released from S+L− cells contain MSV which, upon coinfection with MuLV, is capable of transforming SC-1 cells, a cloned line derived from wild-mouse embryo. This transforming virus is tentatively designated as murine sarcoma virus-zero (MSV-Z). No productive infection can be detected following infection with MSV-Z alone but, in the presence of MuLV, infection of SC-1 cells with MSV-Z results in replication of MSV(MuLV) and MuLV. Exposure of S+L− cells to 5-iododeoxyuridine increases the titer of transforming virus up to 400-fold, but we have detected no comparable increase in reverse transcriptase activity or number of physical particles. No replicating MuLV or xenotropic murine oncornavirus was detected after chemical induction. The peak of transforming activity of MSV-Z is found at a buoyant density 1.15 g/cm 3 in sucrose gradients. MSV-Z cannot be induced from S+L− reverant cell lines. Sensitivity to MSV-Z transformation appears to be limited to the SC-1 cell line although some activity was detected upon infection of NRK cells. Based on glycoprotein interference and neutralization tests, the viral envelope of MSV-Z is distinct from the previously described murine C-type oncornaviruses.

Revertants of mouse cells transformed by murine sarcoma virus. V. Loss of MSV-specific nucleotide sequences from cellular RNA.

Moloney MSV-transformed 3T3FL cells (S+L- cells) gave rise either spontaneously or by chemical induction to flat revertants with in vitro growth properties of nontransformed cells. These flat revertants contained MuLV p30 determinants but did not demonstrate virus production, reverse transcriptase activity, or a rescuable MSV genome. Moloney sarcoma virus- (M-MSV) specific nucleotide sequences were lost from cellular RNA of these cells as determined by nucleic acid hybridization; however, sequences common to M-MSV and M-MuLV viral RNA were observed. In both a phenotypically flat revertant and a rerevertant from which an MSV genome could be rescued, all the viral sequences were transcribed as in the parental S+L- cells. One of the rescue-negative rerevertants failed to demonstrate MuLV p30 determinants, and neither MSV-specific nor common sequences of M-MSV were detected in their cellular RNA as in control 3T3FL Cells. These results indicate that only cells that retained rescuable MSV genomes contained MSV-specific viral RNA, irrespective of the cellular phenotype. By inference, sequences common to M-MSV and M-MuLV viral RNA contained at least part, if not all, of the gag gene. M-MSV-specific nucleotide sequences wete present, however, in DNAs both from revertants and S+L- cells, suggesting that either restriction in transcription of viral genome of in processing of viral RNA sequences is responsible for rescue-negative revertants from S+L- cells.

Revertants of mouse cells transformed by murine sarcoma virus: flat variants without a rescuable sarcoma virus from a clone of BALB/3T3 transformed by Kirsten MSV.

Summary Clonal subline of Kirsten murine sarcoma virus (Ki-MSV)-transformed non-producer BALB/3T3 (Ki-BALB) cells spontaneously produced flat variants with some properties of non-transformed cells at frequencies of 0.01 to 0.001. Such variants were epithelioid, contact-inhibited, grew to low saturation density and exhibited variable cloning efficiences in soft agar. They demonstrated no murine leukaemia group-specific antigen(s), no reverse transcriptase activity, and no infectious virus, but were agglutinable by concanavalin A. Murine leukaemia virus (MuLV) was induced after 5-iododeoxyuridine (IUDR) treatment from both the flat variants and from the parental Ki-BALB cells. However, Ki-MSV could not be rescued from these flat variants by superinfection with MuLV, nor induced by treatment with IUDR. The state of reversion was stable, revealing no back-transformation during 30 to 40 subcultures. The tumourigenicity of the flat variants in BALB/c mice was markedly reduced (actually undetectable) compared to that of the Ki-BALB cells. All flat variants were susceptible to MSV and MuLV infection. The loss of the transformed phenotype in spontaneous flat variants was associated with a decrease in chromosome number to a level similar to BALB/3T3 cells. These observations suggest that these revertants may have lost some or all of the Ki-MSV genome.

Morphologic changes in the rabbit SIRC cell line induced by simian sarcoma-associated virus

Abstract Rabbit cornea (SIRC) cells were altered morphologically upon simian sarcoma-associated virus infection. These focal alterations could be used for virus assay. Unusual small cytoplasmic particles of uncertain origin were observed by electron microscopy in addition to typical C-type particles.

Envelope properties of murine sarcoma virus-zero (MSV-Z)

Abstract Transformation by murine sarcoma virus-zero (MSV-Z), a replication-defective, transforming virus relase from mouse S+L- cells, was interfered with by xenotropic murine leukemia viruses. Host range characteristics and a previously described neutralization pattern of MSV-Z, however, indicate that MSV-Z possesses unique envelope properties which are distinct from the previously recognized murine C-type oncornaviruses.