Specific restriction of avian sarcoma viruses by a line of transformed lymphoid cells.

MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek's disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective (td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supernatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10^?1 to 10^?4 Studies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in both cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virus-specific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fibroblasts. Infectious ASV DNA, while easily detected in fibroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome.     

Viral glycoprotein synthesis under conditions of glucosamine block in cells transformed by avian sarcoma viruses.

The synthesis of the viral glycoprotein gp85 in B77-transformed chick embryo cells cultured under conditions of glucosamine block was studied. As determined by long-term labeling with radioactive fucose, no gp85 is formed in the presence of 20 μmoles/ml of glucosamine, although an intracellular component was detected which was recognized by a serum prepared against gp85 but migrated in SDS-polyacrylamide gel with a greater mobility than gp85. The labeling properties of this component suggest that it is a carbohydrate-deficient form of gp85. These data are discussed in terms of a chemical mechanism for glucosamine block.     

Drug-induced changes of adenylate cyclase activity in cells from asthmatic and nonasthmatic subjects.

Monolayers of adherent mononuclear leukocytes prepared from normal subjects and asthmatic children whose bronchodilater therapy did not include sympathomimetic drugs, respond to relatively low concentrations of isoproterenol (ISO) and prostaglandin E1 with increased intracellular cyclic adenosine monophosphate (cAMP) levels. The magnitude of in vitro responses to ISO is decreased by previous contact of the cells with ISO or other, orally effective, adrenergic drugs. The desensitization is rapid, concentration- and time-dependent, and is readily reversible after removal of the desensitizing drug. The phenomenon exhibits pharmacologic specificity. Cells in which the response to restimulation with ISO was decreased exhibited full sensitivity to prostaglandin E1. No differences in these behaviors were detected in cells from normal or asthmatic subjects. The results suggest that earlier observations reporting decreased responses to beta adrenergic stimulation in asthmatics may have been due to the treatment of these patients with sympathomimetic agents and not caused by a disease-related beta adrenergic receptor dysfunction.     

Cytoskeletal changes induced by two avian sarcoma viruses: UR2 and Rous sarcoma virus

UR2-transformed cells were examined by immunofluorescence and compared to control cells and cells transformed by Rous Sarcoma Virus (RSV). Actin and tubulin which are normally depolymerized in RSV-transformed cells appeared to be unaffected by UR2 transformation. Cell surface fibronectin which is normally lost from RSV-infected cells, appears more abundantly on UR2-transformed cells than on normal cells. Vinculin was shown to be in adhesion plaques in UR2-transformed cells as well as in control fibroblasts but diffuse in the cytoplasm of RSV-transformed cells. Polyacrylamide gel electrophoresis of [35S]methionine-labeled fibronectin and vinculin immunoprecipitated from lysates of normal and transformed cells indicated that cell associated fibronectin synthesized during the labeling period is reduced by 60% in RSV-transformed cells but occurs in normal amounts in UR2-transformed cells. However, immunoprecipitation of radiolabeled fibronectin released in supernatant fluids of normal and transformed cells showed a decreased amount of fibronectin in fluids from UR2-transformed cells, but a considerable increase in the medium from RSV-infected cells as compared to uninfected cultures. These data suggest that more fibronectin binds to the surface of UR2-transformed cells then to normal cells, but is readily released from RSV-transformed cells. Vinculin was reduced by about 50% of normal levels in both RSV- and UR2-transformed cells. Immunofluorescence studies using antibody to virion structural proteins (gag) show that the nuclei of UR2-transformed cells are not fluorescent. This indicates a cytoplasmic location or membrane association for p68ros, the transforming protein of UR2, which contains gag determinants. Overall, these data suggest that changes in the major cytoskeletal proteins of fibroblasts are not essential for the neoplastic properties of cells but are rather a phenotypic expression of transformation, since UR2, which causes tumors in vivo, induces only minor cytoskeletal alterations of cells transformed in vitro.     

Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses

Studies were designed to detect changes in the plasma membrane polypeptide composition of chick embryo fibroblasts (CEF) and Normal rat kidney (NRK) cells following infection with avian RNA tumor viruses. A method capable of isolating large fragments of plasma cell membranes, based on fractionation in a two-phase polymer system, was developed for this purpose.Infection with the Prague (subgroup C) or SR (subgroup A) strains of RSV, or with ts 68 of SR-RSV-A or ts 339 of B77 at the permissive temperature, caused CEF to transform morphologically; simultaneously a limited number of changes occurred in their plasma cell membrane composition. They were: (a) a large increase in the rate of labeling with ¹⁴C-valine and in the total amount of a polypeptide with an apparent MW of 73,000; a similar small increase occurred with respect to a polypeptide with an MW of 95,000; neither could be labeled with ¹⁴C-glucosamine; (b) a decrease in the rate of labeling and in the total amount of high molecular weight glycopoly-peptides with MWs of about 250,000; (c) a decrease in the rate of labeling and in the total amount of a polypeptide with an MW of 39,000. Infection with ts 68 and ts 339 at the nonpermissive temperature, or with a nontransforming mutant of SR-RSV-A, or with RAV-7, did not cause alterations (a) and (b), but did cause alteration (c).Identical membrane polypeptide changes were observed in NRK cells infected with the Prague strain of RSV or with ts 339 of B77 at the permissive but not at the nonpermissive temperature. The significance of the fact that these changes, in particular the increases in the amounts of the 73,000 and 95,000 dalton polypeptides, occurred to the same extent in the membranes of both avian and mammalian cells is discussed.     

Alterations in the genomes of avian sarcoma viruses

We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [³⁵S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.     

Replication of avian sarcoma viruses in chicken macrophages

Avian sarcoma viruses of subgroups B and C were able to replicate in chicken embryonic macrophages (derived from yolk sac) and in adult macrophages (obtained from peripheral blood), while avian sarcoma viruses of subgroups A and D were not. Infectious center assays indicated that the proportion of macrophages infected by viruses representative of subgroups B and C which registered as infectious centers was significantly lower than that of fibroblasts infected by the same viruses. Moreover, the comparative growth of avian sarcoma virus B77 (subgroup C) in fibroblasts and in yolk sac cells showed that macrophages have a reduced ability to replicate this virus. In addition, viruses of subgroups B and C and mainly helper-independent viruses of these two subgroups induced morphological alterations (formation of giant cells) and biochemical changes (increased rate of sugar transport), indicating that the infection of chicken macrophages by avian sarcoma viruses of subgroups B and C led to the malignant transformation of these cells.     

Detection ofBordetella pertussis by determination of adenylate cyclase activity

The adenylate cyclase activity ofBordetella pertussis in clinical isolates was measured in calmodulin-supplemented Stainer-Scholte broth by the rate of conversion of ATP to cyclic AMP. Analysis of 250 stock strains ofBordetella pertussis showed that measurable adenylate cyclase activity was produced by all strains. In clinical testsBordetella pertussis was isolated from 135 (22 %) of 605 swab samples. Increased adenylate cyclase activity was detected in 124 (92 %) Stainer-Scholte broth cultures of these samples. A total of 475 swabs contained other bacteria or had no growth; only one of the Stainer-Scholte broth cultures of these swab samples contained measurable adenylate cyclase activity. The results indicate that testing for adenylate cyclase activity provides a specific and sensitive means for detectingBordetella pertussis in clinical specimens.     

Transformation parameters of chick embryo fibroblasts transformed by Fujinami, PRCII, PRCII-p, and Y73 avian sarcoma viruses

The transformation parameters of chicken embryo fibroblasts transformed by replication defective avian sarcoma viruses were compared with those of cells transformed by Rous sarcoma virus (RSV). Two classes of avian sarcoma virus with transforming genes distinct from the src gene of RSV were examined: Fujinami sarcoma virus (FSV), PRCII, and PRCII-p were used as representatives of one class, and Y73 as a representative of the other class. The transformation parameters examined included morphological markers: cell surface morphology, the disappearance of microfilament bundles, and the loss of cell surface fibronectin; alterations in growth properties: growth to high densities and growth in suspension; and metabolic changes: increased uptake of hexose, release of plasminogen activator, and increased synthesis of hyaluronic acid. In general, all of the viruses induced a phenotype which was qualitatively similar to that of RSV-transformed cells, although the extent of each change was less than that observed with RSV. One strain of FSV was found to be coordinately temperature sensitive for the induction of all phenotypic markers examined, whereas another strain of FSV and the other viruses examined were temperature resistant. PRCII-transformed cells had a lower efficiency of colony formation in agar suspension and showed only partial disruption of microfilament bundles; this partial transformation phenotype may be correlated with the low oncogenicity of this virus. These findings in conjunction with previous studies on protein phosphorylation in ASV-transformed cells are consistent with the idea that the replication-defective avian sarcoma viruses transform cells by a mechanism similar to that involved in transformation by RSV.     

Phenotypic mixing between reticuloendotheliosis virus and avian sarcoma viruses.

Coinfection of chicken embryo fibroblasts with reticuloendotheliosis virus (REV) and avian sarcoma virus leads to the formation of avian sarcoma viral pseudotypes which carry envelope determinants of REV. These pseudotypes can be neutralized by REV antiserum, have a host range which is different from that of any known avian sarcoma virus, and are unable to form foci in cells preinfected with REV. The REV stocks used in these experiments were plaque-purified. They were free of avian leukosis virus detectable in the COFAL tests, and their ability to form pseudotypes with avian sarcoma virus was neutralized with specific REV antiserum.     

Oncogenicity of avian leukosis viruses of different subgroups and of mutants of sarcoma viruses.

Leukosis viruses of seven subgroups were tested for oncogenicity in chickens susceptible to virus infection and to development of lymphoid leukosis (LL) tumors. All subgroup A viruses and the subgroup B virus tested produced a high incidence of LL and other related neoplasms. Viruses of subgroup C and RAV-61 of subgroup F produced a low level of LL. The RAV-50 of subgroup D produced osteopetrosis. In these tests, the viruses of subgroup E and G and one virus of subgroup F were not pathogenic, possibly because infection was not established in the chickens, the chickens were not susceptible to tumor development by these viruses, or the viruses lacked oncogenicity. All temperature-sensitive mutants of Rous sarcoma virus produced sarcomas, but the level varied. One nontransforming mutant produced sarcomas, and the other three tested produced LL. All three mutants that cause cells to grow as colonies in agar produced a high incidence of sarcomas. Thus, sarcoma viruses, by back-mutation, may lose the ability to transform cells in vitro, to make cells grow in agar colonies, or to induce sarcomas in vivo, yet they retain the ability to produce LL. Conversely, it was previously shown that leukosis viruses may be changed into viruses that transform cells in vitro and produce sarcomas in vivo by suitable passage in chicks.     

Temperature-sensitive mutants of avian sarcoma viruses: genetic recombination between multiple or coordinate mutants and avian leukosis viruses.

Five coordinate temperature-sensitive mutants of avian sarcoma viruses which fail to transform or produce infectious progeny at 41° have been analyzed by genetic recombination. Four, namely LA334, 336, 338, and 343, carry multiple mutations. One of these mutations is always in the src gene affecting initiation and maintenance of transformation. The other mutations have not been mapped, but our data suggest that in LA338 there is no linkage of the second mutation with env, whereas in LA343 there is some linkage to env. LA336 has a second mutation affecting an early transient function in accordance with physiological data which have shown a thermolabile polymerase in this virus. The data on LA334 are in accord with previous studies which have indicated lesions in src and gag. For LA337 our data confirm the existence of single coordinate lesion segregating from env.     

Secretin-induced changes in rate,contractility and adenylate cyclase activity in rat heart atria

1. Secretin stimulated adenylate cyclase activity in crude membrane preparations from right and left rat atria, when tested in the presence of the potentiating activator forskolin. Its maximal effect on adenylate cyclase activity was greater in the right atrium than in the left atrium but the peptide efficiency was lower, in both atria, than that ofD, l-isoproterenol.
2. Secretin stimulated the rate of contraction of the spontaneously beating right atrium, but less efficiently thanD,l-isoproterenol. Although this positive chronotropic action of secretin was inhibited byD,l-propranolol, it was probably not mediated by the release of endogenous catecholamines as: a) the inhibitory effects ofD-andL-propranolol were similar and b) the efficiency of secretin on the in vitro beating of right atrium was the same in control and reserpinized rats.
3. Secretin stimulated the force of contraction of spontaneously beating right atria, even in the presence of propranolol, i.e. when the chronotropic effect of secretin was abolished. The hormone exerted also a positive inotropic effect on the electrically stimulated rat atrium. This effect was blocked neither by tetrodotoxin nor by propranolol.
4. When comparing the dose-effect curves of secretin andD,l-isoproterenol on adenylate cyclase activation on the one hand, and on the stimulation of rate and contractility on the other hand, it is tempting to suggest that cyclic AMP might be involved in the modulation by secretin of the mechanical properties of rat atria.