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.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

Comparison of Rous Sarcoma Virus-Specific Deoxyribonucleic Acid Polymerases in Virions of Rous Sarcoma Virus and in Rous Sarcoma Virus-Infected Chicken Cells

Labeled virions of Rous sarcoma virus (RSV) were disrupted with detergent and analyzed on equilibrium sucrose density gradients. A core fraction at a density of approximately 1.24 g/cc contained all of the (3)H-uridine label and about 30% of the (3)H-leucine label from the virions. Endogenous viral deoxyribonucleic acid (DNA) polymerase activity was only found in the same location. Additional ribonucleic acid (RNA)- and DNA-dependent DNA polymerase activities were found at the top of the gradients. RNA-dependent and DNA-dependent DNA polymerase activities were also found in RSV-converted chicken cells. Particles containing these activities were released from cells by detergent and were shown to contain viral RNA. These particles were analyzed on equilibrium sucrose density gradients and were found to have densities different from virion cores.     

Rescue of Rous Sarcoma Virus from Rous Sarcoma Virus-Transformed Mammalian Cells

Rat cells transformed by the B77 strain of avian sarcoma virus produce no virus-like particles, yet B77 virus was rescued from these cells by Sendai virus-mediated fusion with chicken cells. This virus rescue was not affected by treatment of the chicken cells with agents that rendered the cells incapable of dividing, although such treatment greatly reduced the ability of the chicken cells to plate as infectious centers after infection with B77 virus. Fusion of R(B77) cells with chicken erythrocytes also led to virus rescue, although with less efficiency than fusion with chicken fibroblasts. Therefore, virus rescue was probably due to a factor or factors contributed by chicken cells which aid in virus production.     

Molecular events in cells transformed by Rous Sarcoma virus

The Rous sarcoma virus (RSV) transforming gene product has been identified and characterized as a phosphoprotein with a molecular weight of 60,000, denoted pp60src. Partially purified pp60src displays a closely associated phosphotransferase activity with the unusual specificity of phosphorylating tyrosine residues in a variety of proteins. That the enzymatic activity observed is actually encoded by the RSV-transforming gene is indicated by the comparison of the pp60src- protein kinase isolated from cells tranformed by a wild-type RSV or by a RSV temperature-sensitive transformation mutant; these experiments revealed that the latter enzyme had a half-life of 3 min at 41 degrees C, whereas that of the wild-type enzyme was 20 min. Evidence is now beginning to accumulate showing that viral pp60src expresses its protein kinase activity in transformed cells as well as in vitro because at least one cellular protein has been identified as a substrate for this activity of pp60src. Although the protein kinase activity associated with pp60src is itself cyclic AMP (cAMP) independent, the molecule contains at least one serine residue that is directly phosphorylated by the cellular cAMP-dependent protein kinase, thus suggesting that the viral transforming gene product may be regulated indirectly by the level of cAMP. The significance of this latter observation must be regarded from the point of view that the RSV src gene is apparently derived from a normal cellular gene that seemingly expresses in normal uninfected cells a phosphoprotein structurally and functionally closely related to pp60src. This celluar protein, found in all vertebrate species tested, also is a substrate for a cAMP-dependent protein kinase of normal cells, and, therefore, may be evolved to function in a regulatory circuit involving cAMP.     

Nature of Rous sarcoma virus-specific RNA in transformed and revertant field vole cells.

Cytoplasmic and polyribosomal RNAs from Rous sarcoma virus-transformed and phenotypically reverted field vole cells were fractionated by rate-zonal sedimentation and hybridized with a (3)H-labeled complementary DNA viral probe to determine the size classes of virus-specific RNA present in these cell types. In contrast to Rous sarcoma virus-infected permissive avian cells, only two of three discrete species of virus-specific RNA were detected in the cytoplasm of these vole cells. These included genome-length 35S RNA and a 21S RNA. However, viral 28S RNA, routinely detected in the cytoplasm of productively infected avian cells, could not be found in cytoplasmic RNA from vole cells. In addition, a low-molecular-weight viral RNA sedimenting less than 16S was detected in both infected avian and vole cells. Because of its heterogeneity this latter species is most likely generated from the intracellular degradation of the larger viral RNAs. Both the viral 35S and 21S RNA were also found to be associated with total polyribosomes from these vole cells. Studies were also performed to determine the distribution of both total viral genomic and sarcoma-specific RNA sequences among the size classes of fractionated total polyribosomes. In both vole cell types the majority of cytoplasmic viral RNA sequences were also associated with polyribosomes and were similarly distributed among the size classes of total polyribosomes. Sarcoma-specific sequences were present on both the 35S and 21S RNA species. These data suggest that the expression of the viral transforming gene in revertant field vole cells may be controlled at some stage subsequent to translation of the viral RNA.     

New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.     

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.     

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.     

Isolation of virus-specific double-stranded RNA from cells transformed with Rous sarcoma virus

Several methods were used for isolation of double-stranded (ds) RNA from the cytoplasm of Rous sarcoma virus-transformed chick embryo cells. The dsRNA was shown to have a high melting temperature (82.5 degrees C) in 0.16 M phosphate buffer (pH 6.8), which shifted to more than 90 degrees C after RNase treatment. The size of a single strand was approximately 1300-1600 nucleotides and RNase-resistant fragments were 50-250 nucleotides long. Double-stranded RNA formed hybrids with the labeled genomic RSV RNA RNA so that the major subpopulation of the dsRNA hybridized to 6-10% of RSV RNA and the minor subpopulation -- to 90-94% of RSV RNA. It was suggested that this large subpopulation of dsRNA was abundant in sequences homologous to proviral end fragments as judged by Southern procedure. The data are discussed by considering the analogy between retroviral proviruses and mobile genetic elements.     

Neural crest cells: temperature-dependent transformation by Rous sarcoma virus.

Cranial neural crest cells from chick embryos, when cultured under appropriate conditions, differentiate after approx. 1 week into pigmented cells. Neurol crest cells were infested with a mutant (RSV-BH-Ta) of the Bryan 'high titer' strain of Rous sarcoma virus on the second day of culture before the cells were morphologically differentiated, or later after they became pigmented. Cells infected and maintained at the temperature permissive for transformation (37 degrees C) proliferated rapidly compared to uninfected cells are produced extensive cytoplasmic vacuoles in a fashion similar to other types of cells transformed with RSV-BH-Ta at 37 degrees C. Cells infected and maintained at the non-permissive temperature for transformation (41 degrees C) also proliferated rapidly but did not become morphologically transformed. Transformation occurred reversibly following a shift of temperature. Infection of morphologically undifferentiated neural crest cells at either temperature prevented their differentiation into pigment cells, and infection of pigmented neural crest cells at either temperature led to a gradual loss of pigmentation. These results suggest that even at the non-permissive temperature the virus may regulate the state of differentiation of certain types of cells.     

Enzymes and Nucleotides in Virions of Rous Sarcoma Virus

In addition to the previously described deoxyribonucleic acid (DNA) polymerase, DNA ligase, DNA exonuclease, and DNA endonuclease activities, purified virions of Schmidt-Ruppin strain of Rous sarcoma virus (SRV) have nucleotides and nucleotide kinase, phosphatase, hexokinase, and lactate dehydrogenase activities. The SRV virions have no glucose-6-phosphate dehydrogenase activity. All enzyme activities, but glucose-6-phosphate dehydrogenase and adenosine triphosphatase, were increased by disruption of the virions. The DNA polymerase, DNA ligase, and hexokinase activities had a higher specific activity in purified virion cores. It is suggested that during assembly virions of SRV may pick up cytoplasmic components which bind to virion proteins. The role of these components in viral replication is not known at present.     

Properties of enucleated cells. III. Changes in cytoplasmic architecture of enucleated BHK21 cells following trypsinization and replating.

At low concentrations of concanavalin A (conA), binding of the lectin to the erythrocytes appears to be the rate-limiting step in the agglutination of these cells. At higher concentrations of lectin the rate of agglutination is concentration-independent, indicating that the aggregation reaction is rate-determining. Only 5 to 7% of the 1.2 × 10⁵ receptor sites need be occupied by con A in order for agglutination to take place. Although trypsin-treated cells bind 30% less ¹²⁵I-conA, they agglutinate better than untreated cells. At high lectin concentrations, erythrocyte agglutination by wheat germ agglutinin (WGA) is more than 8 times faster than the conA-mediated reaction. Lowering of the temperature to 0 °C reduces the rate but not the extent of the agglutination by both lectins. Mechanical shear reduced the conA-mediated agglutination of native cells by more than 160-fold and that of trypsinized and neuraminidase-treated cells 6-fold and 4-fold, respectively.It is concluded that metabolic activity, receptor mobility (i.e. cluster or patch formation) and cytochalasin B-sensitive processes, all of which have been reported to be involved in the lectin-mediated agglutination of fibroblasts and other cells, do not play a role in erythrocyte agglutination. Lectin-mediated erythrocyte agglutination appears to be governed primarily by the rate and extent of binding of lectin to the cell surface, the cell surface charge (modifiable by enzyme treatments or polycations) and the shear forces in the suspension. Morphological studies confirm and amplify these conclusions.     

Mechanism of Oncogenic Transformation by Rous Sarcoma Virus: I. Intracellular Inactivation of Cell-Transforming Ability of Rous Sarcoma Virus by 5-Bromodeoxyuridine and Light

Chick embryo fibroblasts brought into stationary phase of growth by maintenance in serum-free Eagle's MEM medium were infected with the Bryan strain of Rous sarcoma virus (B-RSV) and incubated for 18 hr in the presence of 5-bromo-deoxyuridine (BUdR). The cells were then allowed to resume growth and deoxyribonucleic acid (DNA) synthesis by addition of an enriched F12 medium containing serum and RSV antibody to prevent spread of viral infection. After 48 hr, the cultures were exposed for various periods to visible light, overlaid with solid culture medium, and observed for the appearance of foci of transformed cells. In cultures treated with BUdR at the time of infection, exposure to light resulted in a suppression of focus formation of from 50 to 90% in various experiments. Treatment with BUdR for 18 hr before infection or on the day after infection, followed by exposure to light, had no effect on focus formation. In cultures in which almost all cells were infected, treatment with BUdR followed by exposure to light did not result in cell death. This suggests that suppression of transformation is not due to selective killing of infected cells by this treatment but rather to the intracellular inactivation of the transforming ability of Rous sarcoma proviral DNA.     

Genetic Determinants of Rous Sarcoma Virus Particle Size

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant ΔNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.     

Triglyceride and cholesterol ester metabolism of rat 1 cells and Rous Sarcoma Virus-transformed rat 1 cells

The incorporation of ¹⁴C-long-chain fatty acids into the neutral lipids of rat 1 cells and B77 Rous Sarcoma Virus-transformed rat 1 cells was compared. Increased labeling of the cholesterol ester and triglyceride fractions was observed in the transformed cells. In temperature shift experiments, using B77 ts src Rous Sarcoma Virus-transformed rat 1 cells, alterations of cholesterol esterification could first be detected 4–6 h after shift to the permissive temperature. Changes in triglyceride metabolism could be shown as early as 4–6 h after activation of the transforming gene function. It is concluded that alterations of neutral lipid metabolism are among the early effects of the transformation process.