Demonstration of avian sarcoma virus-coded pp60src in vivo and the anti-pp60src immune response in chickens

M Ellwart-Tschürtz, R Kurth
1981 Journal of Virology  
The avian sarcoma virus (ASV)-coded transforming protein pp60src was originally detected in vitro in ASV-transformed avian and mammalian cells in experiments involving mammalian antisera to ASV-induced tumors. It is demonstrated here that pp608rc is also expressed in vivo in ASV tumors of chickens. Furthermore, the existence of the endogenous pp6OBarc in all chicken cells does not impair the immune response to exogenous pp6(yrc in the chicken. Whereas chicken antibodies can bind to pp60rc, they
more » ... do not serve as substrates for the protein kinase activity of this transforming protein. The transforming protein of the avian sarcoma viruses (ASVs) is a phosphoprotein of 60,000 molecular weight denoted pp60src. This virus-coded protein is responsible for the in vitro demonstrable malignant transformation of avian fibroblasts into fibrosarcoma cells. In a series of elegant experiments, R. L. Erikson and his collaborators as well as other laboratories have shown that the pp6Osrc molecule is itself phosphorylated and possesses protein kinase activity (for reviews, see references 4 and 7). pp6OBrc was originally demonstrated by precipitation with antisera from tumor-bearing rabbits which reacted with lysates of ASV-transformed chicken cells. Tumor-bearing-rabbit sera were produced by injecting rabbits at birth with high doses of ASV. Subsequent to tumor development and regression, animals were bled repeatedly over a period of 2 to 10 weeks after initial virus injection. The majority of rabbits possessed antibodies which were able to precipitate pp6Osrc from transformed cell lysates. In vitro translation studies have furthermore shown that pp60BrC can be translated from the genome of wild-type ASV, whereas transformation-defective derivatives of these viruses were unable to code for a corresponding molecule. Subsequently, however, a normal cell protein closely related to pp6Osrc was detected in uninfected avian cells as well as in uninfected vertebrate cells. It had a structure and function similar to those of the exogenous pp60r, and it was designated endogenous pp6O arc. There can be little doubt at present that the exogenous pp6O8rc indeed represents the transforming protein of ASVs. One has to keep in mind, however, that all experiments published t Present address: Paul Ehrlich-Institut, 6000 Frankfurt 70, West Germany. to date involve sera from mammals which had been injected either with ASV or with ASVtransformed cells. It is not known whether chickens, the natural host for avian sarcoma leukemia viruses, are able to launch an immune response against pp6)src. The question is whether, due to the presence of endogenous pp6(Oarc in all normal cells, chickens may be tolerant to the pp6Osrc coded for by infectious sarcoma viruses. We were furthermore interested in finding out whether pp6Osrc can indeed be demonstrated in lysates from in vivo tumors, since all published data on the presence of pp6()BC have been obtained with sarcoma cells grown in tissue culture. Two groups, each consisting of six adult line 15 chickens, were injected in the wing web muscle with purified ASV strains Schmidt-Ruppin D or Prague A in increasing concentrations (108 to 1010 focus-forming units). All birds developed palpable fibrosarcomas within 2 weeks. In 8 of the 12 chickens, tumors were subsequently rejected over a period of approximately 4 weeks. The four progressively growing tumors were explanted, and on the same day tumor lysates were prepared and used in classic protein kinase assays with a well-characterized rabbit anti-pp60src serum. Tumor or normal control tissues (1 g) were homogenized in 3 ml of lysis buffer A (175 mM sodium phosphate [pH 7.2], 1% Nonidet P-40, 0.1% deoxycholate, 40 mM NaF, 2 mM phenylmethylsulfonylfluoride, 1% Trasylol) and clarified at 80,000 x gfor 30 min. Increasing amounts of tumor lysate were incubated with 2 ,ul of rabbit anti-pp60src serum for 1 h. The immune complexes were collected with protein A-containing Staphylococcus aureus (6). The precipitates were subsequently washed with buffer B (buffer A containing 0.5 mg of myoglobin per ml), buffer C (buffer A containing 1 M NaCl, 950 on May 9, 2020 by guest
doi:10.1128/jvi.39.3.950-953.1981 fatcat:aqvnb6izgnbvxh34gn72vutadq