Generation of transforming viruses in cultures of chicken fibroblasts infected with an avian leukosis virus

Abstract

During serial passages of an avian leukosis virus (the transformation-defective, src deletion mutant of Bratislava 77 avian sarcoma virus, designated tdB77) in chicken embryo fibroblasts, viruses which transformed chicken embryo fibroblasts in vitro emerged. Chicken embryo fibroblasts infected with these viruses (SK770 and SK780) had a distinctive morphology, formed foci in monolayer cultures, and grew independent of anchorage in semisolid agar. Bone marrow cells were not transformed by these viruses. Another virus (SK790) with similar properties emerged during serial subcultures of chicken embryo fibroblasts after a single infection with tdB77. The 50S to 70S RNAs isolated from these viruses contained a tdB77-sized genome (7.6 kilobases), 8.7-and 5.7-kilobase RNAs, and either a 4.1-kilobase RNA or a 4.6-kilobase RNA. These RNAs did not hybridize with cDNA's representing the src, erb, mac, and myb genes of avian acute transforming viruses. Cells transformed by any one of the SK viruses (SK770, SK780, or SK790) synthesized two novel gag-related polyproteins having molecular weights of 110,000 (p110) and 125,000 (p125). We investigated the compositions of these proteins with monospecific antiviral protein sera. We found that p110 was a gagpol fusion protein which contained antigenic determinants of p19, p27, and reverse transcriptase and that p125 contained only gag determinants, leaving 49,000 daltons which was antigenically unrelated to the structural and replicative proteins of avian leukosis viruses. An analysis of the SK viral RNAs with specific DNA probes indicated that the 5.7-kilobase RNA contained gag sequences but lacked pol sequences and, therefore, probably encoded p125. The transforming ability, the deleted genome, and the induced polyproteins of the SK viruses were reminiscent of the properties of several replication-defective acute transforming viruses.

Activation and transduction of c-mil sequences in chicken neuroretina cells induced to proliferate by infection with avian lymphomatosis virus

“…Molecular probes. The following probes were used: (i) the 1.1-kilobase-pair (kbp) BamHI-HpaI v-mil fragment of the pMH2J3S molecular clone (6); (ii) the 350-base-pair (bp) EcoRI fragment (EcoD) of long terminal repeat (LTR) sequences from the RSV molecular clone pSRA2 (9) (a generous gift of J. M. Bishop); (iii) the EcoRI-BamHI fragment of pLTR-L6 containing U5 and leader sequences of RSV (kindly provided by T. Takeya); (iv) the 2.1-kbp SacI-EcoRI gag-specific fragment of plasmid pB5gag (39); (v) the 1.8kbp KpnI-SacI env-specific fragment of plasmid pB5env (39) (both pB5gag and pB5env plasmids were kindly provided by E. Stavnezer); (vi) a plasmid containing pol sequences obtained by subcloning the 2.2-kb HindIII-KpnI pol-specific fragment of plasmid pSRA2 into pUC18.…”

“…ALVs were also shown to cause tumors by their ability to transduce in vivo cellular oncogenes, including src (17,20), fps (29), erbB (19,28,45), and fos (30). There has been one report of in vitro transduction of a cellular oncogene in chicken fibroblasts chronically infected by a transformation-defective mutant of B77 Rous sarcoma virus (39). Because of the low frequency of these events, early steps of interactions between the ALV genome and regulation of cellular gene expression have been difficult to study in vivo and in vitro.…”

A novel oncogene related to c-mil is transduced in chicken neuroretina cells induced to proliferate by infection with an avian lymphomatosis virus.

“…The following probes were used: (i) the 350 bp EcoRI fragment (EcoD) of long terminal repeat (LTR) sequences from the Rous sarcoma virus molecular clone pSRA2 (DeLozbe et al, 1980; (ii) the 2.1 kb SacI-EcoRI gag-specific fragment of plasmid pB5 gag (Stavnezer et al, 1981); (iii) the 1.8 kb KpnI-SacI env-specific fragment of plasmid pB5 env (Stavnezer et al, 1981); (iv) a plasmid containing pol sequences obtained by subcloning the 2.2 kb HindIII-KpnI pol-specific fragment of plasmid pSRA2 into Transduction of a c-mil-related novel oncogene pUC 18; (v) the 1.1 kb BamHI-HpaI v-mil specific fragment of the pMH2BS molecular clone (Coll et al, 1983).…”

Immunogold labeling of oncogenic and tumor related proteins

“…The apparent paradox of Ski is that it can induce both transformation and myogenic differentiation, seemingly two contradictory properties (Colmenares et al, 1991;Sutrave et al, 1990b). The original v-Ski virus not only caused morphological transformation of chick embryo fibroblasts, but also induced anchorage independent growth of these cells (Stavnezer et al, 1981). In addition, it can induce cells isolated from the body wall of quail embryos to differentiate into muscle (Colmenares and Stavnezer, 1980).…”