Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.     

Phosphorylation and metabolism of the transforming protein of Rous sarcoma virus.

p60src, the transforming protein of Rous sarcoma virus, was found to contain 0.5 to 0.9 mol of total phosphate per mol of polypeptide. The protein is known to be phosphorylated at two sites, a serine in the amino-terminal domain and a tyrosine in the carboxy-terminal domain. Because our indirect analysis suggests that the serine is phosphorylated to approximately twice the extent of the tyrosine, we estimate that p60src contains approximately 0.3 to 0.6 mol of phosphoserine and 0.2 to 0.3 mol of phosphotyrosine per mol of polypeptide. p60src was found to represent approximately 0.02% of the total incorporated radioactivity in Rous sarcoma virus-transformed chick cells labeled with [35S]methionine for 48 h. This corresponds to approximately 500,000 molecules of p60src per cell. Pulse-chase experiments revealed that the half-life of p60src ranged from 2 to 7 h, depending on the strain of virus examined. The P60src of the Schmidt-Ruppin strain was significantly more stable than that of the Prague strain.     

The transforming protein of Moloney murine sarcoma virus is a soluble cytoplasmic protein

The transforming gene, v-mos, of Moloney murine sarcoma virus (M-MuSV) encodes a 37,000-dalton phosphoprotein, p37mos. Since the biochemical function of this protein is unknown, we have determined the subcellular location of p37mos in M-MuSV 124-transformed cells. Using two different methods of cell lysis and fractionation, we found that newly synthesized as well as mature p37mos is a soluble cytoplasmic protein. In agreement with these results, immunofluorescent staining of cells acutely infected with M-MuSV 124, using an antiserum directed against a synthetic v-mos peptide, produced a diffuse cytoplasmic pattern. Gel filtration experiments and glycerol gradient sedimentation analysis suggest that the bulk of p37mos exists as a monomer and is not involved in a specific association with other cellular proteins. These properties of p37mos are different from those of other characterized retroviral transforming proteins.     

A 90,000-dalton binding protein common to both steroid receptors and the Rous sarcoma virus transforming protein, pp60v-src

Previous studies have shown that the avian progesterone receptor, when in the nontransformed 8 S state, is complexed to another cellular protein having a molecular weight of 90,000. In this report, we show that this receptor-binding protein is indistinguishable from the 90,000-dalton protein which associates in a complex with the Rous sarcoma virus transforming protein, pp60v-src. This identity was established by the following criteria. 1) Monoclonal antibodies directed against the pp60v-src-associated 90-kDa protein recognized the 90-kDa progesterone receptor binding protein in an immunoblot assay. Conversely, monoclonal antibodies that recognize the progesterone receptor binding protein bind to the 90-kDa protein which complexes with pp60v-src. 2) Peptide maps prepared from the 90-kDa proteins immunoprecipitated from chicken cells with monoclonal antibodies directed against either the 90-kDa receptor binding protein or the 90-kDa pp60v-src-associated protein were indistinguishable. 3) Preincubation of the progesterone receptor complex with monoclonal antibodies prepared against the pp60v-src-associated protein caused a shift in the sedimentation of the progesterone receptor. Previous studies have established that the pp60v-src-associated protein is indistinguishable from one of the major heat shock proteins which are induced under a variety of stress conditions in eukaryotic cells. These present studies implicate a new role for this 90-kDa protein in the action of steroid hormones.     

It is Rous sarcoma virus protein P12 and not P19 that binds tightly to Rous sarcoma virus RNA

The interactions between Rous Sarcoma virus (RSV) RNA and the viral proteins in the virus have been analysed by Sen & Todaro (1977) using ultraviolet light irradiation; they showed that the major protein ultraviolet light cross-linked to the viral RNA was P19 as identified by polyacrylamide gel electrophoresis. We report here that it is not viral protein P19 but P12 that binds tightly to RSV RNA upon ultraviolet light irradiation of the virus. Therefore, the binding sites of the viral protein along RSV RNA that we have characterized previously should be correctly attributed now to P12 and not P19.     

Cytoskeletal association of pp60src. The transforming protein of the Rous sarcoma virus

Immunoferritin labelling methods have been employed to examine the distribution of the Rous Sarcoma virus (RSV)-transforming protein pp60src in the detergent-resistant cytoskeleton of transformed cells. pp60src was found to be localized on actin microfilaments present in adhesion plaques, at adherens junctions between cells and also in microfilament bundles. This localization is consistent with the hypothesis that some of the morphological effects of transformation result from the interaction in situ of pp60src with microfilament-bound target proteins.     

Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles.

An artificial membrane system was developed to study the molecular basis for interaction of pp60v-src, the Rous sarcoma virus transforming protein, with lipid bilayers. pp60v-src was extracted from cell membranes by detergent solubilization and reincorporated into phospholipid vesicles. Reconstituted pp60v-src retained tyrosine kinase activity and was integrally associated with the liposome through a 10-kilodalton (kDa) amino-terminal domain. The same 10-kDa domain was shown to anchor pp60v-src to the plasma membrane of transformed cells. Reconstitution experiments performed with nonmyristylated pp60v-src proteins revealed that these polypeptides did not interact with phospholipid vesicles. In contrast, myristylated, soluble pp60v-src molecules (including a highly purified pp60v-src preparation) could be reconstituted into liposomes, but their interaction with the liposomal bilayer was not mediated by the 10-kDa amino-terminal domain. When membrane proteins were included during reconstitution of purified pp60v-src, binding through the 10-kDa anchor was restored. A model is presented to accommodate the different types of interactions of pp60v-src with liposomes; the model postulates the existence of an additional membrane component that anchors the pp60v-src polypeptide to the phospholipid bilayer.     

Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus

Vinculin, a protein associated with the cytoplasmic face of the focal adhesion plaques which anchor actin-containing microfilaments to the plasma membrane and attach a cell to the substratum, contains 8-fold more phosphotyrosine in cells transformed by Rous sarcoma virus than in uninfected cells. Because the transforming protein of RSV, p60src, is a protein kinase that modifies cellular proteins through the phosphorylation of tyrosine and because phosphotyrosine is a very rare modified amino acid, this result is a very rare modified amino acid, this result suggests that vinculin is a primary substrate of p60src. Only trace amounts of phosphotyrosine were detected in myosin heavy chains, alpha-actinin, filamin, and the intermediate filament protein vimentin. The modification of vinculin by p60src may be responsible in part for the disruption of the microfilament organization and for the changes in cell shape and adhesiveness which accompany transformation by Rous sarcoma virus.     

The poliovirus receptor protein is produced both as membrane-bound and secreted forms.

Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1----13.2 of human chromosome 19.     

Antiserum specific for the carboxy terminus of the transforming protein of Rous sarcoma virus

An antiserum specific for the carboxy terminus of p60src, the transforming protein of Rous sarcoma virus, was produced by immunization of rabbits with a conjugate of bovine serum albumin and the synthetic peptide NH2-Tyr-Val-Leu-Glu-Val-Ala-Glu-COOH. The carboxy-terminal six amino acids of this peptide correspond in sequence to that deduced for the carboxy terminus of the p60src of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A. The p60src proteins of the several strains of Rous sarcoma virus and the cellular homolog of the viral transforming protein, p60c-src, comprise a polymorphic family of polypeptides. The anticarboxy-terminal serum reacted readily with the p60src proteins of three different strains of Rous sarcoma virus. In contrast, no precipitation of cellular p60c-src could be detected with this serum. This suggests that the viral p60src proteins have identical carboxy termini and that the carboxy terminus of cellular p60c-src may be different from that of viral p60src. The anticarboxy-terminal serum reacted poorly with the subpopulation of viral p60src which is present in a complex with two cellular phosphoproteins. Apparently, the presence of the two cellular proteins interferes with the recognition of p60src by the anticarboxy-terminal serum. It seems likely, therefore, that these two cellular proteins bind to the carboxy-terminal domain of p60src.     

Isolation and partial characterization of a monoclonal antibody to the Rous sarcoma virus transforming protein pp60src.

Transformation of cells by Rous sarcoma virus is mediated by the product of the viral src gene, pp60src. A hybridoma cell line producing an immunoglobulin G3 antibody to pp60src was isolated after lymph node cells from immune mice were fused with mouse myeloma cells (P3-NS1-1). Mice were immunized with p60src purified from Escherichia coli cells expressing the src gene product. The monoclonal antibody immunoprecipitated pp60src from Rous sarcoma virus-transformed cells and recognized an antigenic determinant located in the amino-terminal third of the pp60src protein.     

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

The half-life of metabolically labeled pp60src of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp60src with tumor-bearing rabbit serum. These experiments showed that pp60src has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp60src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42 degrees C) or permissive (35 degrees C) temperature. The half-life of the pp60src -associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp60src. The apparent contradiction between the half-lives of the kinase activity and the [35S]methionine-labeled pp60src protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp60src, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.     

Creation and expression of myristylated forms of Rous sarcoma virus gag protein in mammalian cells.

Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, has long been known to be capable of infecting and transforming mammalian cells; however, such transformed cells do not release virus particles. The RSV gag product (Pr76gag) produced in these cells is not released into the culture medium or proteolytically processed to release mature products. Thus, the behavior of Pr76gag in mammalian cells is much like that of mammalian retroviral Gag proteins which have been altered so as to block the addition of myristic acid at residue 2 (Gly). Because the RSV gag product does not possess a myristic acid addition site, we hypothesized that the creation of one by oligonucleotide-directed mutagenesis might permit particles to be released from mammalian cells. Two myristylated forms of Pr76 were created. In Pr76myr1, the first 10 amino acids have been exchanged for those of p60v-src, which are known to be sufficient for myristylation. In Pr76myr2, the Glu at the second residue has been substituted with Gly. The alleles encoding the modified and wild-type forms of Pr76 have been expressed at high levels in mammalian (CV-1) cells by using an SV40-based vector. Surprisingly, we have found that expression of high levels of the unmodified (wild-type) product, Pr76myr0, results in low levels of particle formation and precursor processing. This indicates that myristic acid is not the sole determinant for targeting. However, the addition of myristic acid to Pr76myr1 or Pr76myr2 resulted in a fivefold enhancement in Gag function. In all aspects examined, the behavior of myristylated Pr76 was identical to that of the authentic product produced in avian cells. We also show that processing is mediated by the gag-encoded protease and that removal of the amino terminus to create Pr76gagX results in an inability to form particles or be processed. This suggests that proper targeting is prerequisite for activation of the RSV protease in mammalian cells.     

Cellular partitioning of beta-1 integrins and their phosphorylated forms is altered after transformation by Rous sarcoma virus or treatment with cytochalasin D.

A sequential extraction procedure of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) buffer followed by RIPA or Laemmli sample buffer was developed to define two distinct subpopulations of beta-1 integrins in primary chicken embryo fibroblasts. Extraction of cells in culture revealed an association of adhesion plaque-localized integrin with the CHAPS-insoluble fraction. Phosphorylated integrins were found in both fractions, but the specific phosphorylation was 12-fold higher in the CHAPS insoluble fraction. The phosphorylation was evenly distributed between phosphoserine and phosphotyrosine. Transformation by Rous sarcoma virus caused a redistribution of integrin to rosettes and an increase in total integrin phosphorylation. Treatment with cytochalasin D caused a redistribution of the adhesion plaque-associated integrin into lacelike structures and reduced the level of integrin phosphorylation. These treatments also caused an altered distribution of phosphorylated integrin between the CHAPS soluble and insoluble fractions. These results suggest a role for integrin phosphorylation in the assembly and disassembly of cellular adhesion structures.     

The membrane-binding domain of the Rous sarcoma virus Gag protein.

The Gag protein of Rous sarcoma virus (RSV) can direct particle assembly and budding at the plasma membrane independently of the other virus-encoded products. A previous deletion analysis has suggested that the first 86 amino acids of RSV Gag constitute a large membrane-binding domain that is absolutely required for these processes. To test this hypothesis, we inserted these residues in place of the N-terminal membrane-binding domain of the pp60v-src, a transforming protein whose biological activity requires plasma membrane localization. The ability of the Src chimera to induce cellular transformation suggests that the RSV sequence indeed contains an independent, functional domain.     

Monomer and multimer covalently closed circular forms of Rous sarcoma virus DNA.

Covalently closed circular molecules of viral DNA synthesized in virus-infected cells are composed mainly of monomers sedimenting at 22 to 27S in neutral sucrose gradients. These monomers are detected by annealing with complementary DNA or transfection assay. However, 11% of the infectious circles sediment faster than monomers. There is a peak at 32S which may correspond to dimer molecules. Traces of infectivity (about 3%) found between 32S and 65S suggest the presence of higher oligomers. In alkaline sucrose gradients, covalently closed monomers are found at 64 to 71S. Infectivity of these monomers is reduced by alkali treatment to less than one-tenth, and, perhaps for this reason, no infectious dimers or higher oligomers are observed. It has been shown that upon resedimentation the dimers of 95 can be separated from monomers and detected by hybridization.     

Two structurally and functionally different forms of the transforming protein of PRC II avian sarcoma virus.

The primary translation product of the PRC II avian sarcoma virus genome is a protein of 105,000 daltons (P105), and we have previously shown that approximately 50% of the P105 molecules are converted to molecules of 110,000 daltons (P110) by posttranslational modification. Fractionation of PRC II-infected cells showed that P105 was contained primarily in a nonionic detergent-soluble compartment, whereas P110 partitioned almost exclusively with a nonionic detergent-insoluble or crude cytoskeletal fraction. The tyrosine-specific protein kinase activity previously observed in immunoprecipitates which presumably contained both P110 and P105 was found predominantly in the P110-containing immunoprecipitates made from the cytoskeletal fraction and was essentially absent from the P105-containing immunoprecipitates prepared from the soluble fraction. Individual analysis of 32P-labeled P110 and P105 prepared by this fractionation technique revealed that P110 contained more phosphotyrosine per mole of protein than did P105. Examination of the tryptic peptide maps of 32P-labeled P110 and P105 suggested that the additional phosphotyrosine in P110 resulted from phosphorylation at discrete sites within the protein. From these experiments, we conclude that PRC II-infected cells contain two discrete forms, P105 and P110, of the transforming protein and that each of these proteins exhibits distinct structural and functional characteristics.     

Structure and self-association of the Rous sarcoma virus capsid protein

BACKGROUND: The capsid protein (CA) of retroviruses, such as Rous sarcoma virus (RSV), consists of two independently folded domains. CA functions as part of a polyprotein during particle assembly and budding and, in addition, forms a shell encapsidating the genomic RNA in the mature, infectious virus. RESULTS: The structures of the N- and C-terminal domains of RSV CA have been determined by X-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy, respectively. The N-terminal domain comprises seven alpha helices and a short beta hairpin at the N terminus. The N-terminal domain associates through a small, tightly packed, twofold symmetric interface within the crystal, different from those previously described for other retroviral CAs. The C-terminal domain is a compact bundle of four alpha helices, although the last few residues are disordered. In dilute solution, RSV CA is predominantly monomeric. We show, however, using electron microscopy, that intact RSV CA can assemble in vitro to form both tubular structures constructed from toroidal oligomers and planar monolayers. Both modes of assembly occur under similar solution conditions, and both sheets and tubes exhibit long-range order. CONCLUSIONS: The tertiary structure of CA is conserved across the major retroviral genera, yet sequence variations are sufficient to cause change in associative behavior. CA forms the exterior shell of the viral core in all mature retroviruses. However, the core morphology differs between viruses. Consistent with this observation, we find that the capsid proteins of RSV and human immunodeficiency virus type 1 exhibit different associative behavior in dilute solution and assemble in vitro into different structures.     

Change of hyaluronic acid synthesis during differentiation of myogenic cells and its relation to transformation of myoblasts by Rous sarcoma virus

Hyaluronic acid synthesis was examined in cultures of differentiating chick embryo muscle cells before, during and after fusion. Prior to fusion, hyaluronic acid was synthesized and secreted into the medium, but once fusion began this synthesis was reduced significantly. Synthesis then increased again after completion of fusion. Thus, production of hyaluronic acid was lowest at the time of or right before cell fusion. When myoblasts were transformed by Rous sarcoma virus (RSV), a higher amount of hyaluronic acid was synthesized, and cells were not able to fuse. The turnover rate of hyaluronic acid might be different between myotubes and RSV-transformed myoblasts. The addition of exogenous hyaluronic acid to myoblast cultures resulted in the partial inhibition of fusion. The effect was reversible because fusion took place after removal of the exogenous hyaluronic acid. These observations suggest that hyaluronic acid plays an important role in the differentiation of myogenic cells, and that elevated hyaluronic acid synthesis may partly be the reason for inhibition of myotube formation upon transformation by Rous sarcoma virus.