Synthesis of Rous sarcoma virus in mammalian embryonal tissue cultures]

In embryonal cells of mice and hamsters growing in vitro after Rous virus adsorption on them and long before the onset of cell transformation of the virus was detected in thr form closely associated with cells. After exposure of mammalian cells to actinomycin D or after lowering the temperature of their incubation up to +20 degrees C the cell associated virus was not revealed in embryonal cells. This evidences that the de novo synthesis of Rous virus but not the survival of the virus takes palce in the mentioned cells.     

Specific increase in polyamine levels in chick embryo cells transformed by Rous sarcoma virus.

Chick embryo fibroblasts and chorioallantoic membranes of chick embryos infected with oncogenic or nononcogenic viruses were analyzed for polyamines. Nononcogenic viruses (influenze, Newcastle disease, or vaccinia virus) had no effect on the polyamine content of chorioallantoic membranes. Transformation of chorioallantoic membranes by a wild-type or temperature-sensitive mutant strain of Rous sarcoma virus under permissive conditions (37 degrees) caused a 2- to 4-fold increase in cellular spermidine and putrescine content. Only putrescine accumulated in chick embryo fibroblasts transformed by Rous sarcoma virus at 37 degrees. At the nonpermissive temperature (42 degrees), the temperature-sensitive mutant, unlike the wild-type strain, did not alter cellular morphology or polyamine content.     

A continuous line of Rous sarcoma virus-transformed chick embryo cells.

A continuous line of Rous sarcoma virus (RSV)-transformed chick embryo cells was established. The cells, designated RTAZ-1, which constitute the only known line of continuously growing RSV-transformed cells of chick embryo origin, grow rapidly, display uniform morphology, and perpetually release large amounts of RSV (Rous-associated virus, type 1). RTAZ-1 cells display a heteroploid chromosome complement with 92-94 chromosomes characteristic of chicken cells. Molecular hybridization studies demonstrated that DNA from these cells hybridized with normal chick DNA but not with human (KB) cell DNA, thus providing additional evidence of the chicken origin of RTAZ-1.     

Pathogenesis of Rous sarcoma virus in the chick embryo with particular reference to vascular lesions

1. Growth of strains of Rous sarcoma virus on the chorio-allantoic membrane continues throughout the embryo's development, with some decrease in the rate of growth at the end. Susceptibility of the chorio-allantoic membrane, as measured by the total number of lesions following a standard inoculum, was greatest at 11 to 13 days of incubation. The proportion of vascular pocks of the membrane was however much higher in embryos inoculated at 7 or 8 days of incubation.

• 1 Growth of virus in the chick embryo liver after intravenous inoculation was also continuous throughout incubation. Age of susceptibility, again measured by number of lesions, was however greatest at 8 to 10 days.
• 2 Examination of vascular lesions in the living embryo, and study of whole mounts stained by periodic acid-Schiff method as well as of histological sections, all indicated that the vascular lesion was at least predominantly a localized neoplasm of the vascular endothelium. The early presence of PAS-positive cells in the developing pock was demonstrated, an observation hitherto limited to tissue culture.
• 4 The presence of antibody to Rous virus in the yolk of the embryo was correlated with a decrease of vascular lesions following inoculation of 13-day embryos but not ofj I-day embryos.
• 5 Following intravenous inoculation, virus was probably limited to the initial area of infection during the first few days, but viremia was irregularly demonstrable from 3 days on.
• 6 In the discussion it is emphasized that tumors of the embryo or the chicken provoked by the Rous virus are primarily tumors of tissues derived from the mesoderm. This includes the capacity to cause vascular lesions. The production of avion lymphomatosis may well be an exception, but since the lymphatic tissue has not been clearly demonstrated to be of ectodermal origin, this remains an open question. Virus growth and tissue culture ‘ transformation ’ do occur in a variety of tissues.

     

Selective effect of Rous sarcoma virus src gene expression on contractile protein synthesis in chick embryo myotubes

Myogenic precursor cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and maintained at the permissive temperature for transformation. Following subculture, a population of cells was produced which failed to differentiate at the permissive temperature but produced a high proportion of myotubes in sister cultures shifted to the nonpermissive temperature. The myotube-containing cultures were further enriched for this cell type by the addition of 1-beta-D-arabinofuranosylcytosine to kill replicating mononucleated cells. This myotube population was suitable for testing the effect of viral-transforming gene expression in a postmitotic, terminally differentiated cell which expresses relatively low levels of the cellular homologue of the viral-transforming gene and is resistant to infection by the virus. The consequence of shifting these Rous sarcoma virus-infected myotube cultures to the permissive temperature was assessed in terms of protein synthesis. The total rate of incorporation of exogenous radioactive leucine, supplied at a concentration which saturated the intracellular pool, was similar between cultures held at the two temperatures, suggesting that the absolute rate of total protein synthesis was not affected by expression of viral-transforming gene. In contrast, the rate of synthesis of eight proteins the expression of which is specific for myotubes was suppressed reversibly. The rate of synthesis of five other proteins which are not selectively concentrated in myotubes was either unaffected or stimulated. Thus. the expression of viral-transforming gene in myotubes leads to differential effects on developmentally regulated proteins without inducing several properties of the transformed state classically observed for fibroblastic cells.     

Decrease in collagen production in normal and Rous sarcoma virus-transformed chick embryo fibroblasts induced by phorbol myristate acetate.

The effect of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on collagen synthesis, a differentiated property of chick embryo fibroblasts, was examined. Collagen synthesis, as measured by the rate of formation of [3H]hydroxyproline from [3H]proline, was found to be decreased in cells treated with PMA but not in cells treated with the parent alcohol phorbol. The decrease in collagenase-sensitive proteins was confirmed by polyacrylamide gel electrophoresis of cell lysates, indicating that the decrease could not be ascribed simply to an effect on prolyl hydroxylase. Although a decrease in collagen synthesis was observed after one day, five days were required for a maximal reduction to 20% of that of dimethyl sulfoxide-treated controls. The effect of PMA on collagen synthesis was reversible. It was therefore not the result of a permanent transformation of the cells or of the selection of a population of cells with a reduced capacity for collagen synthesis. Collagen synthesis was decreased in chick embryo fibroblasts transformed by Rous sarcoma virus. Treatment of these cells with PMA for 5 days brought about a further decrease to 50% of the level in dimethyl sulfoxide-treated transformed controls.     

A variant Schmidt-Ruppin strain of Rous sarcoma virus with increased affinity for mammalian cells

SR-RSV-D(H), a variant virus with extremely high tropism for mammalian cells, was isolated by passage of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup D (SR-RSV-D) through hamster cells. This variant virus has acquired an altered envelope glycoprotein, encoded by the env gene, that has high affinity for receptors on the surface of mammalian cells. The variant virus transforms rat cells at about 100 times the efficiency of the parental virus, SR-RSV-D(S), as assayed by focus formation. Addition of amphotericin B (Fungizone) to the medium at a concentration of 0.2 micrograms/ml completely inhibited rat cell transformation by SR-RSV-D(H), possibly by blocking virus penetration into the cells, whereas the drug showed no inhibitory effect on transformation of chick embryo fibroblast (CEF) cells by the variant virus or on transformation of rat cells by the parental virus. The efficiency of transformation of rat cells by the variant virus was much less than its efficiency of transformation of CEF cells. Analysis of infection of rat cells suggested that the virus can infect rat cells as efficiently as CEF cells but that rat cells were not transformed by the virus as fully as CEF cells because of inefficiency of some post-penetrational step involved in viral gene expression. The finding that E1AY cells, rat cells expressing adenovirus E1A gene, were transformed by SR-RSV-D(H) as efficiently as CEF cells supports this conclusion and suggests that expression of the E1A gene in rat cells may overcome the defect in the transforming step(s) in rat cells.     

Rous sarcoma recurrence and Rous sarcoma virus growth in chicken muscle

Following Rous sarcoma virus (RSV-Bryan standard strain) infection and tumor regression in chickens, tumors recurred in 3 of 6 chickens after contralateral inoculation of 10⁵ of RSV. In an attempt to understand the mechanism of tumor recurrence, chickens which had recovered from RSV infection were inoculated at the regression site with Rous associated virus (RAV). Tumors recurred in one-third of those chickens that received three bi-weekly RAV injections and in one chicken that received only a single RAV injection. The tumors re-appeared from 6 to 19 weeks after regression of the original tumors and within 4 weeks of the last RAV challenge. RSV was recovered from cultures of a recurring tumor as well as from a wing that developed subcutaneous blistering following RAV inoculations. RSV was also obtained from 40% of “recovered” wing muscles explanted in tissue culture up to 3 months after regression. In addition, RSV was isolated from a tumor that recurred in one of 12 recovered chickens inoculated with Freund's adjuvant. In an attempt to determine if ageing of muscle contributed to resistance to infection and suppression of tumor recurrence, wings of 5-week-old and 12-week-old chickens were infected with RSV and RAV, or RAV alone. RSV and RAV were recovered from most muscles explanted 48 h or more after RSV infection of 5-week-old chickens. However, when muscles from 12-week-old chickens were explanted 4 days after infection, very little RSV was recovered, although most of the RAV-infected wings produced RAV in culture. This suggests that RAV growth in older muscles is probably not the limiting factor in RSV growth. The relationship between various factors involved in resistance to tumor development and tumor recurrence in chickens recovering from RSV infections is discussed.     

Expression of viral protein P27 in avian sarcoma virus-transformed mammalian cells and helper-dependent rescue of Rous sarcoma virus.

Avian sarcoma virus (ASV)-transformed mammalian cells, known to be either virus-producing, virogenic or non-virogenic, were tested for viral gene products: for p27 by radioimmunoassay and for group-specific (gs) antigens by the complement fixation test. Clones of B77 virus-producing RBI rat tumour cells had p27 concentrations ranging from 10 to 80 ng/mg cellular protein; the poor virus-producing clone had the lowest levels of p27 and gs antigen. Treatment of the clones with halogenated pyrimidines, BrdUrd and IdUrd, had little effect. Virogenic mammalian cell lines XC, LWC3 cells and human 118MG/EH cells transformed by the Prague, Schmitt-Ruppin and Engelbreth-Holm strains of ASV, respectively, were analyzed for p27, for gs antigens, tumour induction in chickens and focus formation after fusion of the X-ray-irradiated cells with chicken fibroblasts. Concentrations of p27 and of gs antigens and tumour induction by intact cells were approximately the same in both rat XC cells and human 118MG/EH cells. Treatment with BrUrd or IdUrd increased the level of p27 in the latter cell line. On the other hand, rat LWV3 cells had no detectable p27 or gs antigens but their tumourinducing activity in chickens was fifty-fold higher. Non-virogenic rat TWERC cells, transformed by the Prague strain of RSV, had low detectable levels of p27 and of gs antigens. These cells did not produce tumours in chickens or foci when fused with chicken fibroblasts. The only, and very effective, way of rescuing RSV from TWERC cells was the fusion of TWERC cells with RAV-1-preinfected cells. After inoculation of a mixture of 2×10⁴ cells treated with inactivated Sendai virus, tumours were induced in 50% of the chickens. Both the host-range and virus neutralization studies indicated that the rescued TWERC virus had avian subgroup C specificity and not the specificity of the helper virus. The rescued virus had a one-hit titration pattern in duck embryo fibroblasts, indicating that the virus was non-defective for infection and transformation of these cells.