Partial Transcription of Murine Type C Viral Genomes in BALB/c Cell Lines

The mouse cell line, BALB/c 3T3, and its derivatives transformed either spontaneously or by treatment with a variety of external agents, were analyzed for cytoplasmic RNA complementary to DNA products prepared from the Kirsten strain of murine sarcoma-leukemia virus, and from an endogenous type C virus of BALB/c 3T3. Although none of these cell lines spontaneously releases complete type C virions, they all contain RNA which is partially homologous to a portion of the 35S RNA isolated from these viruses. The parental cell line, BALB/c 3T3, contains a low level of viral-related RNA, and there is an increased amount of this RNA in some of the transformed cells. The RNA detected represents only a fraction of the viral RNA found in virus-producing cells. The formation of RNA:DNA hybrids was detected by equilibrium centrifugation in Cs(2)SO(4) density gradients and by analysis with a single-strand-specific nuclease from Aspergillus oryzae. Viral DNA products prepared either from an endogenous reaction with whole virus in the presence of actinomycin D or from purified 70S viral RNA as template using avian myeloblastosis virus DNA polymerase yield comparable data. In addition, all of the BALB/c lines examined produce detectable levels of murine type C virus group-specific antigen.     

Selection of Revertants of Kirsten Sarcoma Virus Transformed Nonproducer BALB/3T3 Cells

Revertants of Kirsten sarcoma virus transformed nonproducer BALB/3T3 cells (KA31 cells) were isolated after exposing the transformed cells to 5-fluorodeoxyuridine at high cell density, or when suspended in methylcellulose. Revertants were also isolated by treating KA31 cells with the lectin, concanavalin A, which is manyfold more toxic to transformed cells than for normal cells. The revertants resemble BALB/3T3 cells in their morphology and growth characteristics in that they have a low saturation density, fail to grow in 1% calf serum or when suspended in methylcellulose, and cease to synthesize DNA after reaching their saturation density. Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus. The concanavalin A revertants also become transformed after infection with murine leukemia virus. All the revertants can be transformed by Kirsten sarcoma virus but not by simian virus 40.     

No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus.

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were estimated by the method of fiber-autoradiography (J. A. Huberman and A. D. Riggs, J. Mol. Biol.32:327-341, 1968) and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcoma virus-transformed BALB/c 3T3 cells.     

Nucleic Acid Homology of Murine Type-C Viral Genes

The nucleic acid sequence homology between various murine, endogenous type-C viruses (three host range classes of BALB/c virus, the AT-124 virus, and the CCL 52 virus) and two laboratory strains of murine leukemia virus (Rauscher and Kirsten) was determined by DNA:RNA hybridization. The viral sequences exhibit varying degrees of partial homology. DNA:DNA hybridizations were performed between [³H]DNA probes prepared from N- and X-tropic BALB/c endogenous viruses and cellular DNAs from BALB/c, NIH Swiss, and AKR inbred mouse strains as well as from California feral mice and the Asian mouse subspecies Mus musculus molossinus and M. musculus castaneus. All of these strains of mice are shown to possess multiple (six to seven per haploid genome), partially related copies of type-C virogenes in their DNAs. Thermal melting profiles of the DNA:RNA and DNA:DNA hybrids suggest that the partial homology of the viral nucleic acid sequences is the result of base alterations throughout the viral genomes, rather than the loss of discrete segments of viral sequences.     

Synthesis of type C virus particles from murine-cultured cells induced by iododeoxyuridine. V. Effect of interferon and its interaction with dexamethasone.

Previous studies have shown that in certain cell systems dexamethasone may enhance the production of type C viruses. Conversely, interferon has been shown to inhibit their production. Both appear to exert their influence late in the viral replication cycle rather than on the synthesis of viral-specific RNA. In this report dexamethasone and interferon have been used to study some aspects of the mechanisms involved in the synthesis of type C viruses in murine K-BALB cells following induction of virus production by iododeoxyuridine. Interferon inhibited production of xenotropic type C virus induced by iododeoxyuridine from K-BALB cells both in the absence and presence of dexamethasone, but it did not affect production of N-tropic type C virus. Exposure of the cells to interferon for longer than 12 h was required for maximum effect. Two types of inhibitory effects were observed: one diminished by dexamethasone when the steroid was added 24 h after interferon removal, and the second resistant to dexamethasone. The concentration of intracellular group-specific antigen was diminshed after interferon and increased after dexamethasone exposure. When induced cells were treated with both interferon and dexamethasone, the intracellular group-specific protein concentration was slightly increased, but virus production was reduced 10-fold compared with induced cells treated with dexamethasone alone. We conclude that interferon and dexamethasone may affect both the synthesis of viral proteins and the assembly or release of virus particles and that dexamethasone can partially nullify the inhibitory activity of interferon. The results also support previous conclusions that the regulatory mechanisms for synthesis of viral proteins and for the release of viral particles may differ and that controls for xenotropic and ecotropic virus formation may not be identical.     

Release of xenotropic type C RNA virus in response to lipopolysaccharide: acitivity of lipid-A portion upon B lymphocytes.

The present studies demonstrate that lipopolysaccharide (LPS) causes the release of endogenous xenotropic type-C RNA virus from BALB/c spleen cells. The evidence suggests that virus release is stimulated by the lipid-A portion of LPS and primarily involves an action of LPS on B lymphocytes. LPS had little or no effect on virus release by T lymphocytes, macrophages, or fibroblasts. These results indicate that the differentiated state of the cell plays an important role in the regulation of endogenous virus release.     

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.     

Structure and Functions of the Kirsten Murine Sarcoma Virus Genome: Molecular Cloning of Biologically Active Kirsten Murine Sarcoma Virus DNA

The unintegrated closed circular form of viral DNA prepared from NIH3T3 cells infected with Kirsten murine sarcoma virus was cloned into bacterial plasmid pBR322. The closed circular DNA, which consisted of two different-sized populations, was enriched from the virus-infected cells, linearized with BamHI, and inserted into pBR322 DNA. Four different recombinant DNAs (clones 2, 4, 6, and 7) were obtained, and a physical map of each was constructed by using various restriction enzymes. Clone 4 DNA had the largest insertion, corresponding to a complete copy of the linear DNA. This suggested that this insertion contained two copies of the 0.55-kilobase pair long terminal redundant sequence. Clone 2 and clone 6 insertion DNAs had deletions of 0.2 and 0.5 kilobase pair, respectively, which mapped near the right end (3' side of viral RNA) of the linear DNA. Clone 7 DNA appeared to have a deletion of a single copy of the large terminal redundant sequence. Transfection of BALB3T3 cells with the clone 4 DNA insertion showed that this DNA had transforming activity. The efficiency of transfection with clone 4 Kirsten murine sarcoma virus DNA was enhanced eightfold by inserting EcoRI-cleaved viral DNA into the EcoRI site of pBR322. The EcoRI-inserted DNA produced foci with single-hit kinetics, suggesting that a single molecule of Kirsten murine sarcoma virus DNA can induce transformation. Results of transfections with EcoRI-inserted Kirsten murine sarcoma virus DNA cleaved with various restriction enzymes suggested that the first 3.3-kilobase pair region at the left end of the linear DNA is important for the initiation of transformation or maintenance of transformation or both.     

Isolation of highly fusogenic variants of simian virus 5 from persistently infected cells that produce and respond to interferon.

A series of experiments were undertaken to examine how interferon and neutralizing antibodies influence the ability of simian virus 5 (SV5) (strain W3) to establish and maintain persistent infections in murine cells. In contrast to the rapid decline in SV5 protein synthesis observed in murine BALB/c fibroblasts (BF cells), which produce and respond to interferon, between 24 and 48 h postinfection there was no inhibition of virus protein synthesis in MSFI- cells, skin fibroblasts derived from alpha/beta-interferon receptor knockout BALB/c mice. Furthermore, the addition of anti-interferon antibodies to the culture medium of infected BF cells significantly reduced the observed decline in virus protein synthesis. Following infection of untreated BF cells, the majority replicated virus but survived the infection and eventually cleared the virus after 8 to 15 days. However, not all the cells were cured, and the cultures became persistently infected. Upon passage of persistently infected cultures, the virus fluxed between active and repressed states as a consequence of interferon production. This resulted in a balance being reached in which only 5 to 20% of the cells were infected at any one time. After 30 passages of the persistently infected cells, highly fusogenic virus variants arose (one of which was isolated and termed W3-f). W3-f remained as sensitive to interferon as the parental W3 isolate but, in the absence of interferon, spread much more rapidly than the parental W3 strain through BF cell monolayers. Sequence analysis revealed no deduced amino acid differences between the F proteins of W3 and W3-f. BF cell cultures persistently infected with W3-f were rapidly cleared of virus by the addition of virus-neutralizing antibodies to the culture medium. In contrast, neutralizing antibodies had little effect on the numbers of cells persistently infected with W3 over several passages. These results suggest that the ability of paramyxoviruses to cause cell-cell fusion may be selected for in vivo as a consequence of their adaptation to the interferon response rather than their need to escape from neutralizing antibodies. The significance of these observations with regard to persistent parainfluenza virus infections in vivo is further discussed.     

Cyclic AMP-independent processes mediate Kirsten sarcoma virus-induced changes in collagen production and other properties of cultured cells

Previous studies suggested that the decreased collagen production observed in Kirsten sarcoma virus (Ki-MSV)-transformed BALB 3T3 cells could be reversed by treating cells with Bt2cAMP. We examined the relationship between intracellular cAMP, collagen production, and other properties in NRK and BALB 3T3 cells transformed by Ki-MSV. Two 3T3 transformants (Ki-3T3-234 and Ki-3T3Cl1) had lower cAMP levels than nontransformed cells. The level in a temperature-sensitive transformant, tsKi-3T3-714, was similar to the level in its parent, 3T3-714, and when it was shifted to a temperature nonpermissive for transformation (40 degrees C), intracellular cAMP did not increase although the growth and morphological properties were normal. The relative rate of collagen production also increased to the normal level. These results indicate that transformation-induced changes were regulated independently of cAMP. Further observations supported this conclusion. Intracellular cAMP in a flat revertant of Ki-3T3Cl1 was lower, rather than higher, than in the transformant, although the relative rate of collagen production was higher. Treatment of Ki-3T3-234 and tsKi-3T3-714 with cholera toxin plus isobutylmethylxanthine increased intracellular cAMP concentrations to 2-20 times the level in untreated cells, levels much higher than in nontransformed 3T3. In spite of this, collagen production was not increased by these agents in tsKi-3T3-714 and it was only partially restored in Ki-3T3-234 relative to the level in the nontransformed cells. In contrast, these agents inhibited growth on a substratum or in soft agar and produced a flattened morphology in both lines. Similarly, collagen production in transformed NRK cells (K-NRK) was only 3% of normal but treatment with Bt2cAMP or cholera toxin plus isobutylmethylxanthine increased production to only one-third the normal level while increasing cAMP to four times the normal level. We conclude that in Ki-MSV-transformed BALB 3T3 cells, changes in cAMP may be secondary effects and not related to maintenance of the transformed phenotype. The high levels of cAMP induced by exogenous agents may act on similar targets as those affected by transformation, but reversal of the transformed phenotype by these agents probably occurs by a different mechanism than that originally used to impose the changes.     

Selective inhibition of cell growth and associated changes in glycolipid metabolism induced by monovalent antibodies to glycolipids

Monovalent antibodies directed against N-acetylhematoside are growth inhibitory for BALB/3T3 and NIL hamster fibroblasts but not their transformed counterparts. Within a similar dose range antibodies directed against globoside have no effect on cell growth. Inhibition of 3T3 cell growth by anti-hematoside correlates with a specific change in the metabolism of hematoside within the cell membrane. Following antibody treatment the radiolabeling of hematoside is elevated for cell in logarithmic growth but reduced relative to control at final saturation density. This effect is not observed for 3T3 cells transformed by Kirsten murine sarcoma virus. It is suggested that cell surface glycolipids may play a role in the control of normal cell growth in vitro.     

Cell surface glycolipids of transformed NIH 3T3 cells transfected with DNAs of human bladder and lung carcinomas.

Neutral glycolipids and gangliosides of NIH 3T3 cells oncogenically transformed by transfection of DNAs from human lung carcinoma (Lx-1) and human bladder carcinoma (Ej) have been investigated. The chemical quantity and the degree of cell surface exposure of gangliotriaosylceramide (Gg3) were greatly enhanced in NIH 3T3 cells transformed by transfection of DNAs of either Lx-1 or Ej carcinoma cells. An identical but more conspicuous change in cell surface exposure of Gg3 was observed in BALB/c 3T3 cells transformed by murine sarcoma virus Kirsten strain, but the same glycolipid was absent in the original Lx-1 or Ej human carcinomas. The mechanism that defines the chemical quantity and the organization of glycolipids is controlled by multiple factors. These include not only the quantity but also the organization of glycosyl transferases and hydrolases in membranes. This also involves membrane dynamics regulated through a cytoskeletal-membrane conjunction which may determine the degree of glycolipid exposure at the cell surface. The similarity of the chemical and organizational change of a single glycolipid, Gg3, between 3T3 transformants by Kirsten murine sarcoma virus and those by transfection of human cancer DNAs may indicate a common biochemical basis triggered by activation of the oncogene.     

Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture.

To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells. Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions. In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX. Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity. Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone. However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator. The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells.     

Induction of cytopathogenicity in mammalian cell lines challenged with culturable enteric viruses and its enhancement by 5-iododeoxyuridine

Cultures of 17 established cell lines were tested against 105 enteric virus types for capacity to support viral replication as indicated by cytopathogenic effect production. Enhancement of susceptibility by treatment of the cells with 5-iododeoxyuridine was evaluated in parallel with untreated cells. Cytopathogenic effect was produced in two or more cell lines by every virus tested except six strains of group A coxsackie virus. No cell line was found to be susceptible to these six virus types. In general, treatment with 5-iododeoxyuridine provided a more rapid onset of cytopathogenic effect in susceptible cells and in some instances resulted in refractory cells becoming permissive to viral replication. The use of 5-iododeoxyuridine allowed two human embryonic lines (HEL-299 and L-132), in combination, to be susceptible to all but the six group A coxsackie virus strains.     

Juvenile hormones inhibit murine cell cycle progression and expression of type c viruses

Summary Juvenile hormones (JH), congeners of retinoic acid, were examined for their capacity to inhibit cell cycle progression and chemically induced expression of endogenous xenotropic retrovirus in Kirsten sarcoma virus-transformed BALB (K-BALB) mouse cells. JHI, II, and III were found to inhibit induction of virus by 5-iododeoxyuridine (IUdR) and histidinol (Hdl) in a concentration-dependent fashion. Some inhibition of macromolecular synthesis was observed upon culture of the cells with JH; the most affected was RNA synthesis, which was reduced 27 to 40% within 4 h by the juvenoids. Epoxide hydrase (EH) activity, as determined by high-pressure liquid chromatography (HPLC), was present in amounts sufficient for the cells to convert the hormones metabolically to an ultimate form. A contact-inhibited K-BALB variant was synchronized by mitotic arrest and the cell cyclespecific effect of JHIII on virus induction during S phase was studied. JHIII added during G₁ phase, and followed by induction, inhibited virus expression 95 and 76% by IUdR and Hdl, respectively. Induction was inhibited only 35% when JHIII was added during S phase concomitantly with the inducers and no inhibition was observed when JHIII was added during G₂ phase followed by the inducers. JHIII added to synchronous cells in G₁ phase inhibited progression of cells into S phase and the onset of DNa synthesis. The results indicate that mouse fibroblasts have a juvenile hormone-sensitive restriction point in G₁ phase that might relate to the effects these hormones have on cell replication and differentiation. This work was supported under Contract NO-1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205.