Association of gag-myc proteins from avian myelocytomatosis virus wild-type and mutants with chromatin.

Abstract

The localization of the transformation‐specific proteins was analyzed in quail embryo fibroblast cell lines transformed by wild‐type avian myelocytomatosis virus MC29 and by three of its deletion mutants, Q10A, Q10C, and Q10H, with altered transforming capacities, and in a chicken fibroblast cell line transformed by the avian erythroblastosis virus (AEV). These viruses code for polyproteins consisting of part of the gag gene and of a transformation‐specific region, myc for MC29 and erb A for AEV. Analysis by indirect immunofluorescence using monoclonal antibodies against p19, the N‐terminal region of the polyprotein, showed that the gag‐myc proteins in cells transformed by the wild‐type MC29 as well as by the three deletion mutants are located in the nucleus. In contrast, cells transformed by AEV, which express the gag‐erb A protein, give rise to cytoplasmic fluorescence. Fractionation of cells into nuclear and cytoplasmic fractions and analysis by immunoprecipitation and gel electrophoresis confirmed these results. About 60% of the gag‐myc proteins of wild‐type as well as of mutant origin were found in the nucleus, while 90% of the gag‐erb A protein was present in the cytoplasm. Also, pulse‐chase analysis indicated that the gag‐myc protein rapidly accumulates in the nucleus in just 30 min. Further, it was shown that the wild‐type and also mutant gag‐myc proteins are associated with isolated chromatin. Association to chromatin was also observed for the gag‐myc protein from MC29‐transformed bone marrow cells, which are believed to be the target cells for MC29 virus in vivo.

Decreased DNA-binding ability of purified transformation-specific proteins from deletion mutants of the acute avian leukemia virus MC29.

“…This shows that the DNA binding is not a consequence of a particular established cell line but is a property in common with the protein synthesized in the actual target bone marrow cells. As described before, the v-myc proteins in three quail fibroblast cell lines transformed by three nonconditional mutants of MC29 were also located in the nucleus, as was shown by immunofluorescence analysis, and were found to be associated with chromatin (11). However, we found here that the three purified mutant v-myc proteins had decreased DNA-binding abilities and that two of them lost their specificities to bind to poly-(dG)poly(dT).…”

“…DISCUSSION The putative transforming protein of avian myeloblastosis virus MC29, p1109a9-mYc, is a nuclear protein, as was shown by immunofluorescence analysis with specific antibodies and by cell fractionation (4,10,11). Moreover, it is a DNA-binding protein (4) that is tightly associated with chromatin (11). The data presented here give evidence that a direct correlation exists between the potential of transformation of hematopoietic cells by MC29 and the DNA-binding properties of the v-myc protein.…”

“…A similar observation was made with mutant Q1OH (data not shown). DISCUSSION The putative transforming protein of avian myeloblastosis virus MC29, p1109a9-mYc, is a nuclear protein, as was shown by immunofluorescence analysis with specific antibodies and by cell fractionation (4,10,11). Moreover, it is a DNA-binding protein (4) that is tightly associated with chromatin (11).…”

“…The role of these two proteins is still unclear. Fluorescence microscopy of AEV-transformed nonproducer chicken fibroblasts with monoclonal or rabbit antibodies against p19 gave rise to cytoplasmic fluorescence with no indication of a distinct nuclear fluorescence in contrast to MC29-transformed quail fibroblasts (10,11). The third gag-fusion protein analyzed here was that of Fujinami sarcoma virus (FSV)-transformed rat cells (12).…”

“…Additional evidence for the specificity of the p1109as-rYc-IDNA interaction is given by the complete lack of DNA binding by p75ga9rbA, one of the two putative AEV-transforming proteins (9), and by p130(t54is, the transformation-specific protein from FSV (12). Both proteins were shown to be absent from the nucleus of transformed cells (10,11,18). Although nothing is known about the possible transformation mechanism by AEV, due to the insufficient knowledge about the second putative transforming protein p61erbB (9), a kinase has been found to be associated with P130oag-fPs from FSV-transformed cells (13), even after purification (ref.…”