Intracellular Viral RNA Species in Mouse Cells Nonproductively Transformed by the Murine Sarcoma Virus

The size and quantity of virus-specific RNA in five non-virus-producing mouse cells transformed by the Moloney isolate of murine sarcoma virus (MSV) was determined. Hybridization of RNA from transformed cells with the [(3)H]DNA product of the RNA-directed DNA polymerase of the murine sarcoma-leukemia virus was used to detect and quantitate virus-specific RNA. The amount of virus-specific RNA in non-virus-producing cells was less than one-sixth of that found in virus-producing cells. A striking correlation was found between the amount of intracellular virus-specific RNA and the degree of agglutination by conconavalin A previously reported for the four non-virus-producing NIH/3T3 cell lines (Salzberg and Green, 1974). A major RNA subunit sedimenting at 26 to 28S was detected in all five MSV-transformed non-virus-producing cells. This could represent the RNA genome of defective MSV.     

Expression of a New Complement-fixing Antigen reactive with Murine Sarcoma Virus Rat Antiserum in Rat Cells transformed by Polyoma Virus

WE reported accelerated transformation by DNA viruses (SV40 and polyoma) in rat embryo (RE) cells chronically infected with a C-type RNA virus^1,2. Recently we found in RE cells transformed by polyoma virus a new complement-fixing (CF) antigen detectable by rat antisera having broad reactivity with the various intraspecies and interspecies antigens of the RNA tumour viruses^3–8; this antigen, however, was distinct from the murine intraspecies and interspecies group-specific (gs) antigens both immunologically and by virtue of other properties. It is also distinct from the polyoma virion (capsid) and tumour (“T”) antigens.     

Rat C-Type Virus induced in Rat Sarcoma Cells by 5-Bromodeoxyuridine

HALOGENATED derivatives of uridine are highly effective inducers of latent C-type RNA viruses^1,2 and have been successfully used to induce viruses identical to, or similar to, the C-type RNA tumour viruses in mouse, rat and human cells^3–6. In previous experiments we used 5-bromodeoxyuridine (BrUdR) for induction of focus-forming virus in non-productive rat cells that have been transformed by mouse sarcoma virus². We describe here the induction of a C-type RNA virus in the cells of the rat tumour cell line XC, which contains the Rous sarcoma virus genome⁷. The induced virus possesses the group specific (gs) antigens of rat C-type viruses but not those of chicken C-type viruses.     

Changes in the cell surface architecture in normal and polyoma virus transformed hamster cells after infection with influenza virus

Test of Con A induced cell agglutination, method of binding cells to Con A coated nylon fibres and modified procedure of cell-to-cell binding were used in the investigation of architectural surface changes in normal and polyoma virus transformed hamster cells infected with influenza virus. In both cell types influenza virus infection caused 1) increase in fixation resistant Con A agglutination, 2) decrease in the level of surface membrane fluidity and cell plasticity. It has postulated that influenza virus infection results in stabilization of the cell surface architecture. These changes are amplified by polyoma virus transformation. Con A acts in this system, as an indicator rather than as a modifier of architectural changes.     

Biochemical characterization of cells transformed via transfection by feline sarcoma virus proviral DNA.

Murine fibroblasts transformed by transfection with DNA from mink cells infected with the Snyder-Theilen strain of feline sarcoma virus and subgroup B feline leukemia virus were analyzed for the presence of integrated proviral DNA and the expression of feline leukemia virus- and feline sarcoma virus-specific proteins. The transformed murine cells harbored at least one intact feline sarcoma virus provirus, but did not contain feline leukemia virus provirus. The transformed murine cells expressed an 85,000-dalton protein that was precipitated by antisera directed against feline leukemia virus p12, p15, and p30 proteins. No feline oncornavirus-associated cell membrane antigen reactivity was detected on the surfaces of the transformed murine cells by indirect membrane immunofluorescence techniques. The 85,000-dalton feline sarcoma virus-specific protein was also found in feline cells transformed by transfection. However, these cells also contained env gene products. The results of this study demonstrate that the feline sarcoma virus genome is sufficient to transform murine cells and that expression of the 85,000-dalton gag-x protein is associated with transformation of both murine and feline cells transformed by transfection.     

Infection of Tumour Cells by Fowl Plague Virus in the Presence of Actinomycin D

INFLUENZA virus is one of the few viruses in which replication is inhibited by the antimetabolite actinomycin D (AM-D)^1–4, which inhibits DNA-dependent RNA synthesis in mammalian cells⁵. It has been reported that the growth of fowl plague virus (FPV) in virus-transformed hamster cells is less sensitive to AM-D^6,7. We have examined the sensitivity of FPV to AM-D to see whether it is related to differences in the oncogenic properties of tumour cells. We found that in cells transformed by polyoma virus (PV) and also in cells transformed by methylcholanthrene, although no infectious virus was produced the cells synthesized viral haemagglutinin (HA). It was only the cell-associated HA, however, that was affected by AM-D and not that released by the cells.     

Activation of the Murine Sarcoma Virus Genome After Infection with the Murine Leukemia Virus as Determined by Cell Agglutination

Non-virus-producing NIH/3T3 cells transformed by the murine sarcoma virus are agglutinated by conconavalin A to the same low level as normal NIH/3T3 cells. Infection with the murine leukemia virus greatly increases the agglutination of transformed cells but not that of normal cells. These data suggest that the morphological expression of cell transformation and the surface alterations associated with increased cell agglutination are controlled by the expressions of different sarcoma virus genes.     

Nature of Ribonuclease-resistant Nucleic Acid of Chick Embryo Cells transformed by Schmidt–Ruppin Strain of Rous Sarcoma Virus

THE mode of replication of RNA or RNA-containing tumour viruses is not understood. The recent studies on Rous sarcoma and other RNA-containing oncogenic viruses suggest that the replicative cycle of the RNA of these viruses might not be associated with ribonuclease-resistant structures (double stranded RNAs), but might involve the synthesis of a DNA intermediate specific to viral RNA^1–3. Two groups of workers, however, presented evidence for the presence of a double stranded RNA in 78 Al cell line of rat embryo fibroblasts which had been transformed and chronically infected with the murine sarcoma-leukaemia virus complex (MSV-MLV)^4,5 and it was suggested that the mode of replication of oncogenic viral RNAs was the same as that of non-oncogenic viral RNAs⁴. This apparent discrepancy prompted me to look for ribonuclease-resistant RNA structures in the chick embryo cells transformed by Schmidt-Ruppin Rous sarcoma virus (SR-RSV).     

Inhibitor of the DNA-dependent DNA Polymerase of Some RNA Tumour Viruses in Feline Sera

AN inhibitor of the RNA-dependent DNA polymerases^1,2 of mammalian C-type viruses was found in sera from rats bearing transplantable tumours, induced by murine C-type RNA tumour viruses^3,4. Partially purified polymerases of murine leukaemia virus³ and feline leukaemia virus (FeLV)⁴ were shown to be antigenic in rabbits and a rat, respectively. We have detected an inhibitor of the DNA-dependent DNA polymerase^5,6 of feline and murine C-type viruses in the sera of cats inoculated in utero and/or postnatally with the Gardner-Arnstein strain of feline sarcoma virus (FSV)⁷ and in the sera of cats bearing spontaneous sarcomas, lymphomas or carcinomas.     

Feline Leukaemia Viral Antigens and Antisera to the Group Specific Antigens of the Murine Leukaemia Viruses

GEERING et al.¹ reported that feline leukaemia viruses shared one of the group specific antigens of the murine leukaemia viruses, gs-3, as detected by immunoprecipitation in agar gels with broadly reactive rat antisera to the group specific antigens of the murine leukaemia viruses (MuLV). Subsequently, they found that this shared group specific antigen was also present in the hamster and rat C-type viruses². Work by Schafer³ and our own immunodiffusion⁴ and complement fixation studies have confirmed the immunological reactivity between the feline leukaemia viral antigens and broad-reacting murine leukaemia group specific antisera. We have now applied this interspecies immunological reaction between the murine and feline C-type viruses to quantitative studies of the feline leukaemia viruses. Broad-reactive murine leukaemia-sarcoma group specific antisera prepared in rats by the induction of murine sarcoma virus (MSV) tumours^{5, 6} were found to be as useful and nearly as sensitive as a feline leukaemia-sarcoma specific, group specific antiserum for the in vitro detection and assay of the noncytopathogenic feline leukaemia virus (FeLV).     

Cyclic amp-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus: Expression of transformation-associated markers

Normal rat kidney (NRK) cells infected with a temperature-sensitive (ts) mutant of mouse sarcoma virus (NRK [MSV-1b]) express the transformed phenotype when grown under permissive conditions, but acquire the normal phenotype when grown under restrictive conditions. Addition of 3', 5' cyclic adenosine monophosphate (cAMP) to NRK (MSV-1b) cells grown at the restrictive temperature results in morphological transformation. To determine whether other markers associated with the transformed phenotype were coordinately expressed after cAMP exposure, concanavalin A (Con A) agglutinability, hexose transport rate, and incorporation of radioactively labeled fucose into fucolipid III and fucolipid IV (FL III and FL IV ) of the cells were examined. NRK cells transformed by wild-type MSV or NRK(MSV- 1b) grown under permissive conditions were agglutinated by low concentrations of Con A and exhibited relatively high maximal agglutination levels which were specifically inhibited by α-methyl-D-mannoside. In contrast, NRK (MSV-1b) cells grown under restrictive conditions were weakly agglutinated by Con A and exhibited reduced maximal agglutination levels, similar to uninfected NRK cells. Treatment of NRK (MSV-1b) cells at the restrictive temperature with cAMP resulted in morphological transformation and a change in the pattern of incorporation of labeled fucose inot FL III and FL IV to one comparable to that of NRK (MSV-1b) cells at the permissive temperature or to NRK cells transformed by wild-type MSV. In contrast, cAMP treatment resulted in no increase in Con A agglutinability or 2 deoxy-D- [(3)H]glucose transport relative to mock treated cultures. The results demonstrate that cAMP-induced morphological transformation and altered fucolipid composition of NRK (MSV-1b) cells are not correlated with alterations in hexose transport rate or Con A agglutinability.     

Agglutination of Normal Cells by Plant Lectins following Infection with Nononcogenic Viruses

MAMMALIAN cells transformed by oncogenic viruses and chemical carcinogens undergo characteristic changes in their surface properties, some of which affect the control of cell multiplication. Certain plant lectins agglutinate transformed cells but not normal cells^1–6, which, although possessing binding sites, can only be agglutinated following treatment with proteolytic enzymes^3–5. Furthermore, both normal and transformed cells bind equal amounts of lectins, indicating that the increased susceptibility of transformed and trypsinized cells to agglutination is not caused by simple “unmasking” of hidden receptor sites. Nevertheless, the increased susceptibility of normal cells to agglutination following trypsinization may well result from changes occurring in the cell coat material. Since lytic infections with certain nononcogenic viruses¹⁰ and various drug treatments¹¹ are known to cause modification of the coat material in normal cells, we were interested to see whether these treatments increased the susceptibility of cells to agglutination by lectins.     

Spontaneous Release of Endogenous Ecotropic Type C Virus from Rat Embryo Cultures

Type C viruses were isolated from embryo cultures of two different rat strains, Sprague-Dawley and Fischer. Both viruses (termed rat leukemia virus, RaLV) were released spontaneously from rat embryo cells, have a density of 1.14 to 1.15 g/cm(3) based on equilibrium sedimentation in sucrose gradients, contain 60-70S RNA, RNA-directed DNA polymerase, and rat type C virus-specific 30,000 molecular-weight-protein determinants. Molecular hybridization studies using the Sprague-Dawley RaLV 60-70S RNA show that the virus-specific nucleotide sequences are present in the DNA of rat embryos. Both Sprague-Dawley and Fischer RaLV can rescue the murine sarcoma virus genome from Kirsten murine sarcoma virus-transformed nonproducer cells and are neutralized by antisera to the RPL strain of RaLV. In contrast to previous RaLV's, these viruses propagate in their own cells of origin as well as in cells of heterologous rat strains.     

Agglutination of Normal and Rous Sarcoma Virus-transformed Chick Embryo Cells by Concanavalin A and Wheat Germ Agglutinin

RODENT cells in culture transformed by oncogenic DNA viruses have surface sites that on normal cells are usually present in latent form only^1,2. This difference in surface properties can be detected by plant glycoproteins such as wheat germ agglutinin (WGA) and concanavalin A (Con A), which agglutinate only transformed cells, because they have certain carbohydrate moieties on their neoplastic surfaces^1–4. According to some investigators, normal and neoplastic cells that have been freshly isolated also exhibit this marked difference^3,5; according to others^6,7, there is no such distinction. We have looked for such differences in cells transformed by RNA tumour viruses and in several types of normal and naturally occurring malignant cells and their normal counterparts.     

Size of virus-specific RNA in B-34, a hamster tumor cell producing nucleic acids of type C viruses from three species.

B-34 is the designation of a hamster tumor-derived cell line induced by the Harvey sarcoma virus. This cell line produces virions which contain structural proteins common to edogenous hamster viruses and nucleic acid sequences of hamster, mouse, and rat origin. The sedimentation characteristics of the intracellular virus-specific RNA was determined in sucrose gradients after treatment with dimethylsulfoxide by molecular hybridization using complementary DNA of strict virus specificity. Hamster virus-specific RNA sedimented at 35S (major peak) as is characteristic of productive infection by type C leukemia viruses of other species. Rat virus-specific RNA sedimented at 30S which is characteristic of the sarcoma virus-related genome found in nonproducer cells transformed by Kirsten sarcoma virus. Both Harvey and Kirsten sarcoma viruses contain a related but not necessarily identical 30S rat-specific component which is also found in normal cultured rat cells. Mouse cells producing Harvey sarcoma virus also contain a rat-specific 30S RNA. Mouse virus-derived sequences also sedimented at 30S in B-34 cells and in a similar size range in Harvey virus-infected mouse cells. The possibility that the mouse and rat-derived sequences are present on a single 30S RNA species which would then be related to sarcomagenic potential is one attractive hypothesis suggested by these data.     

Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.     

Inhibition of spontaneous aggregation by wheat germ agglutinin in suspensions of BALB/c-3T3 and BHK21 tissue culture fibroblasts

A quantitative method of measuring cytoaggregation based on the Coulter electronic cell counter has been applied to the agglutination of BALB/c-3T3 and BHK21 tissue culture fibroblasts by wheat germ agglutinin. When agglutinin is added to transformed or trypsinized cell suspensions high aggregation rates are seen, and the results obtained are in close agreement with the well-known sensitivity of transformed cells to agglutination by lectins.In the absence of agglutinin, a small but reproducible amount of spontaneous aggregation can also be detected. It is related in some way to growth, since it is absent in suspension prepared from confluent (density-inhibited) cultures and is induced by either transfer to low density, additional serum, or transformation by viruses. Under conditions favouring growth, both BALB/c-3T3 and BHK21 cells show aggregation indices close to 25, corresponding to 10% of the maximum aggregation rate seen.This spontaneous aggregation is susceptible to inhibition by agglutinin. Inhibition occurs at low concentration (about 10 μg/ml) and aggregation rates thus pass through a minimum as the concentration of agglutinin is increased. Among the four different cell lines studied, sensitivity to inhibition is inversely related to agglutination. Thus 3T3 cells, which are barely agglutinated by 1 mg/ml of agglutinin, show 90% inhibition; polyoma virus-transformed BHK cells, which are agglutinated by 10 μg of agglutinin, show no inhibition at all.It is suggested that the agglutination of transformed cells is a consequence of their failure to respond to an inhibitory effect exerted by lectins upon an intrinsic adhesive property of the cell surface.     

Induction of Long Term Lymphocyte Lines from Delayed Hypersensitive Human Donors using Specific Antigen plus Epstein–Barr Virus

THE availability of homogeneous populations of human and murine myeloma cells has provided a unique opportunity for investigating the mechanism of immunoglobulin formation¹. Continuous lines of cultured lymphoid cells producing specific antibody or manifesting delayed hypersensitivity would be even more useful in studying the molecular events of the immune response. Human lymphoid cell lines have been established in long term culture using Epstein–Barr virus (EBV)^{2, 3} or phyto-haemagglutinin⁴ but antigen alone has not been effective⁵. The purpose of the work reported here was selectively to establish antigen-sensitive cells in culture by stimulating peripheral white cells from delayed hypersensitive donors with antigen in vitro and then exposing the cells to EBV. This combination of antigen and virus was chosen because of the following considerations: (1) some RNA and DNA viruses do not replicate in resting lymphocytes but can infect antigen-sensitive lymphocytes which have been stimulated in vitro with mitogens or specific antigen^{6, 7}; (2) polyoma virus transforms cells in the G₂ phase of the cell cycle more effectively than in G₁ (ref. 8). These observations suggested that combined exposure to antigen and EBV might result in the establishment of cell lines enriched for antigen-sensitive or antibody-forming cells.     

Binding of Radioactively Labelled Concanavalin A and Wheat Germ Agglutinin to Normal and Virus-transformed Cells

VARIOUS substances isolated from plants cause animal cells to clump. Several of these lectins¹ preferentially agglutinate cells which have been transformed spontaneously or by chemicals or viruses^2–7. The best known lectins of this class are concanavalin A (Con A) isolated from jack beans⁸ and wheat germ agglutinin⁴, which seem to bind to carbohydrate groups on the cell surface. The determinants recognized by the lectins seem to be N-acetyl-D-glucosamine for WGA⁴ and probably α-methyl-D-glucopyranoside for Con A⁶.