Membrane lipid acyl group alterations in cells infected with a temperature-conditional mutant of Rous sarcoma virus

The increased percentage of oleic acid and decreased percentage of arachidonic acid which occurs in the lipids of chicken emrbyo fibroblasts transformed by Rous sarcoma virus was shown to be transformation specific rather than a consequence of virus infection. Cells infected with a temperature conditional mutant of Rous sarcoma virus (RSV-T5) has a normal type fatty acid composition when held at the restrictive temperature of 41°C, but had a transformed type fatty acid composition when held at the permissive temperature of 36°C. However, these acyl group changes occurred slowly in the course of transformation, suggesting that they are not a primary event in the genesis of the transformed phenotype.     

Alterations in lipid acyl group composition and membrane structure in cells transformed by Rous sarcoma virus.

The acyl group composition of the phospholipids from normal chick embryo fibroblasts and from cells transformed by Rous sarcoma virus was determined by gas-liquid chromatography. Rous-transformed cells had less arachidonate (20:4) and more oleate (18:1) in membrane lipids than normal, growing cells. Normal density-inhibited cells had the lowest ratio of 18:1/20:4. Associated with the decreased content of 20:4 in the transformed cells was a decreased motional freedom of an incorporated spin-labeled fatty acid analogue. Arrhenius plots for uptake of 2-deoxyglucose revealed an increased apparent activation energy in the transformed cells, suggesting that the hexose transport carriers were sensitive to the changes in membrane composition and structure in fully transformed cells. However, the development of the changes in fatty acid composition occurred relatively slowly in the course of transformation, indicating that the observed compositional alterations are not likely to be a primary cause of the early changes in membrane function associated with malignant transformation.     

Dissociation of transformation parameters using temperature-conditional mutants of Rous sarcoma virus.

tsGI251 is a mutant of Rous sarcoma virus which induces a partially transformed phenotype at 42°C (Becker et al., 1977). We have considered two alternative explanations for the partial transformation properties of tsGI251: the src gene product could have more than one intracellular target, or tsGI251 could be a “leaky” mutant. To test the second hypothesis, we cultured cells infected with a variety of temperature-conditional transformation mutants at temperatures between the restrictive and permissive temperatures to make them “leak” to varying degrees, and then measured the following parameters of transformation: growth in soft agar, growth in the absence of serum, density-dependent growth inhibition, LETS protein on the surface, hexose transport rate, plasminogen activator activity and adhesion to the culture dish. We found that the properties of tsGI251 could not be mimicked by making any of the other mutants “leak.” Moreover, although all the parameters of transformation varied coordinately with temperature in most of the mutant-infected cells, cells infected with tsGI251, while thermo-sensitive for cellular parameters of transformation, were cold-sensitive for growth. This indicates that tsGI251 is not simply a “leaky” mutant and raises the possibility that src has more than one primary intracellular target. In addition, the ability of temperature-conditional mutants to cause a dissociation of transformation parameters provides a useful tool for analyzing cause-effect relationships in transformation.     

Role of cell division in differentiation of myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus.

The relationship between a potential requirement for cell DNA synthesis and the expression of differentiated muscle cell functions was investigated using primary chicken embryo myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV). Under optimized conditions, transformed myoblasts growing at the permissive temperature could differentiate into multinucleated myotubes, express muscle-specific myosin, desmin and acetylcholine receptors in the absence of DNA synthesis and cell division following a shift to the non-permissive temperature. Furthermore, the experiments demonstrate that individual RSV-infected myoblasts have two options: either to divide and express the transformed phenotype or to withdraw from the cell cycle and differentiate into myotubes. The choice between these options appears to depend on the protein-kinase activity of pp60src, the src gene product.     

Recombination between a temperature-sensitive mutant and a deletion mutant of Rous sarcoma virus.

Cells doubly infected with two mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), ts68, which is temperature sensitive for cell transformation (srcts), and a deletion mutant, N8, which is deficient in the envelope glycoprotein (env-), produced a recombinant which carried the defects of both parents. The frequency of formation of such a recombinant was exceptionally high and made up 45 to 55% of the progeny carrying the srcts marker. By contrast, the reciprocal recombinant, which is wild type in transformation (srcts) and contains the subgroup A envelope glycoprotein (envA), was almost undetectable. This remarkable difference in the frequency of the formation of the two possible recombinants suggests that a unique mechanism may be involved in the genetic interaction of the two virus genomes, one of which has a large deletion. When an RNA-dependent DNA polymerase-negative variant of the N8 (N8alpha) was crinants also became deficient in the polymerase. Cells infected by the srctsenv- recombinant were morphologically normal at the nonpermissive temperature (41 degrees C) and susceptible to all subgroups of RSV. The rate by which the wild-type RSV transformed the recombinant-preinfected cells was indistinguishable from that of transformation of uninfected chicken cells by the same wild-type virus. This indicates that no detectable interference exists at postpenetration stages between the preinfected and superinfecting virus genomes and confirms that the expression of the transformed state is dominant over the suppressed state.     

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 degrees C), while numerous large aggregates of particles are present at the non-permissive temperature (41 degrees C). Changes in the distribution of particles after cells are shifted from 36 to 41 degrees C are observed after 20 min, while a temperature shift from 41 to 26 degrees C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41 degrees C and from 41 to 36 degrees D do not require protein synthesis or RNA synthesis.     

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

Summary Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions, from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.     

Transfer of defective avian tumor virus genomes by a Rous sarcoma virus RNA packaging mutant.

SE21Q1b, a Rous sarcoma virus mutant which packages cellular rather than viral RNA, is competent for infection of quail cells and can transmit defective transforming retrovirus genes. Stably transformed recipient clones have been obtained by using this mutant.     

Transformation-dependent alterations in the oligosaccharides of Prague C Rous sarcoma virus glycoproteins.

The influence of cell transformation on the glycosylation of viral envelope glycoproteins was examined by high-resolution gel filtration and specific glycosidase digestions of 3H-sugar-labeled glycopeptides from nondefective and transformation-defective Prague C strains of Rous sarcoma virus replicated in fibroblasts from the same chicken embryo. The major difference in glycosylation attributable to the viral transformation of the host cells was an increase in this relative amount of larger acidic-type oligosaccharides containing additional "branch" sugars (NeuNAc-Gal-GlcNAc-) compared with the smaller acidic-type and neutral-type oligosaccharides. There was also a shift in size distribution of neutral-type oligosaccharides toward smaller oligomannosyl cores in the transforming versus nontransforming virus glycopeptides. These alterations were consistent with a transformation-dependent increase in the extent of intracellular processing of a common precursor structure for the asparagine-linked oligosaccharides of Rous sarcoma virus.     

Phosphotyrosine-containing proteins and expression of transformation parameters in cells infected with partial transformation mutants of Rous sarcoma virus.

We have examined the phosphorylation state of five proteins known to become phosphorylated on tyrosine during transformation by Rous sarcoma virus by using cells infected with a panel of partially transforming mutant viruses. Situations of viral mutant and growth temperature were found in which phosphorylation of some proteins occurred more extensively than that of others, indicating that mutations in the src gene had affected the specificity of pp60src for some of its substrates as well as affecting the activity of the enzyme. To obtain insight into the biological functions of these phosphorylations, comparisons were made between the degree of phosphorylation of these proteins and the expression of various indicators of the transformed phenotype. The data suggest that phosphorylation of proteins l, p, and q (Mr of 46,000, 39,000 and 28,000, respectively) is not sufficient to induce changes in adhesiveness, hexose transport or morphology. The phosphorylation of protein p or l or total phosphotyrosine content correlated well with the production of plasminogen activator, and the phosphorylation of proteins l and q correlated well with increased hexose transport. However, even when good correlations were observed, significant exceptions were sometimes noted. It thus remains possible that some phosphorylations on tyrosine observed in Rous sarcoma virus-transformed cells are not causally related to the expression of the measured parameters of transformation.     

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.     

Transformation of chicken embryo retinal melanoblasts by a temperature-sensitive mutant of Rous sarcoma virus.

Retinal melanoblasts were transformed by a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). At the permissive temperature for transformation, the cells cease melanin synthesis, degrade their melanosomes and release much of their accumulated melanin into the medium. At the nonpermissive temperature, the cells assume an epithelioid morphology, actively synthesize melanin and become difficult to distinguish from normal uninfected control cultures. Both the transformed phenotype and the differentiated cell phenotype are temperature-dependent. Infected retinal melanoblasts which are incubated at the nonpermissive temperature and which accumulate a large amount of melanin are unable to transform in response to a temperature shift; instead, the cells degenerate and die. Retinal melanoblasts can be infected by subgroups A, B, C and D of RSV; however, their level of susceptibility to infection is about 1/40 compared to fibroblasts. Cultures infected by ts-RSV produce virus at both temperatures, suggesting that cell phenotype does not regulate virus synthesis.     

Decreased rate of S-adenosyl-L-homocysteine metabolism: an early event related to transformation in cells infected with Rous sarcoma virus.

An increase of methylase activity is often related to neoplastic transformation. SAH, the natural inhibitor of transmethylases, does not inhibit cell transformation induced by RSV, in contrast to one of its synthetic analogues, SIBA. This inefficiency was thought to be due to the rapid metabolism of SAH by transformed cells. We now show, that, on the contrary, 70 % of the added amount of SAH disappears in one hour in cell-free extracts of normal cell against only 14 % in extracts of transformed cells. This decreased rate of degradation occurred one day post infection. Cells infected with the non transforming RAV₁ degrade SAH at the same rate as normal cells. A decrease of SAH-hydrolase and adenosine deaminase activity was also observed in infected cells. The decrease of the first enzyme seems to be related to the transformed state, whereas that of the second enzyme seems to depend only on infection, since it is also observed in cells infected with RAV₁.