The defective acute leukemia viruses avian myeloblastosis virus (AMV) and E26 virus each contain an inserted cellular sequence related to the same highly conserved cellular gene, proto-amv. The oncogenes of these two retroviruses differ from this cellular proto-oncogene in gene structure, transcript structure, and gene product. AMV (7,8).In leukemic myeloblasts, the AMV oncogene is expressed as a 2-kb spliced subgenomic mRNA (9, 10). In contrast, proto-amv is expressed as a 4-kb transcript in normal and certain neoplastic hematopoietic tissues (11,12). This protoamv mRNA contains both 5' and 3' genetic information not present in AMV (ref. 13; unpublished data). In addition to differing gene and transcript structure, differing protein products of the AMV oncogene and proto-oncogene have also been found (3, 4).By using three antisera raised against different synthetic peptides predicted from the amv open reading frame, a common 48,000 Mr protein was specifically identified as the AMV oncogene product, p48mY', in AMV-transformed leukemic myeloblasts (3). The same three antisera also specifically precipitated a 110,000 Mr candidate normal cellular homolog, p110Protoa'v, in hematopoietic tissues that express the 4-kb proto-amv message (3).As a first approach to analyzing the functions of the AMV oncogene product, we have determined its-intracellular location. In addition, we have determined the location of the putative transforming protein, p135sag8""I'ts, of the E26 leukemia virus, which contains an oncogene with both amv-related and -unrelated cellular sequences (ets) (14-17). Leghorn chicken embryos (Pacesetter, Anaheim, CA). All cells except for the RP-9 cell line were cultured as described (3). RP-9 was maintained in liquid culture in the same media but supplemented with 20%o heat-inactivated chicken serum and 10% fetal calf serum. Avian erythroblastosis virus (AEV) was kindly provided by P. Duesberg (Molecular Biology Laboratory, University of California, Berkeley). MATERIALS AND METHODSCell Labeling. 4265The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Two cellular proteins (molecular weights: 56,000 and 32,000) were specifically co-immunoprecipitated by simian virus 40 and BK virus t-antigens and by polyoma virus non-T early proteins.In a previous study (20), we demonstrated that two cellular proteins with apparent molecular weights of 56,000 (56K) and 32K were specifically iunmunoprecipitated in association with the simian virus 40 (SV40) t-antigen. The 56K and 32K proteins were found in infected and in uninfected cells. To demonstrate the presence of the proteins in uninfected cells, extracts of radiolabeled uninfected cells were mixed with unlabeled extracts of SV40-infected cells before immunoprecipitation. The specificity of the interaction was shown by the use of SV40 deletion mutants that do not synthesize intact small tantigen and by the use of specific antisera (10). The 56K and 32K proteins were not present in immunoprecipitates of cells infected with t-antigen deletion mutants or when antisera that do not recognize the viral t-antigen were used for immunoprecipitation.Other transforming papovaviruses code for the synthesis of t-antigens that resemble the SV40 t-antigen both in size and in sequences of cysteine-rich regions of the proteins (2, 3, 17). Consequently, it was of interest to determine whether t-antigens of the human papovavirus BK virus (BKV) and the mouse virus polyoma could promote the co-immunoprecipitation of cellular proteins from infected cells.Immunoprecipitation of proteins from BKVinfected cells was studied by using human embryonic kidney (HEK) cells that had been infected with wild-type BKV for 72 h at 37°C (8,19). The infected cells were pulsed with [35S]methionine for 24 h, then extracted with 0.5% Nonidet P-40 in Tris-buffered saline, pH 8. Extraction buffers included 2 to 5 mM dithiothreitol. Immunoprecipitation was done according to standard procedures (20). Proteins were immunoprecipitated by using antisera from hamsters bearing tumors of the SV40-transformed cell line, THE-2 (10). The SV40 and BKV large Tand small t-antigens cross-react immunologically (11,15), and the antitumor sera raised against THE-2 cells interacted with these proteins at high efficiency. Antisera raised in animals bearing BKV-induced tumors were not used because all sera tested in these laboratories lacked detectable antibody to viral small t-antigens and recognized only large T-antigens of SV40 and BKV.Immunoprecipitates of BKV-infected HEK cells contained the viral proteins, T and t (21K), but also contained two proteins with apparent molecular weights of 56K and 32K (Fig. 1B). Electrophoretically similar 56K and 32K proteins were also present in immunoprecipitates of SV40-infected monkey kidney (CV-1) cells (Fig. 1C), but not in immunoprecipitates of cells infected with SV40 deletion mutants that lacked t-antigen (Fig. 1D). To determine whether the 56K and 32K proteins observed in immunoprecipitates of BKV-infected cells were cellular in origin, mixed extract experiments were done. Uninfected CV-1 cells were labeled with methionine and then mixe...

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Nuclei obtained from chicken leukemic myeloblasts transformed by avian myeloblastosis virus were fractionated into various subnuclear compartments, which were then analyzed by specific immunoprecipitation for the presence of the leukemogenic product, p48v-myb, of the viral oncogene. In cells labeled for 30 or 60 min with L-[35S]methionine and in unlabeled exponentially dividing leukemic cells analyzed by Western blotting, p48v-myb was detected within the nucleoplasm (29 +/- 9% [standard deviation] of the total), chromatin (7 +/- 4%), and lamina-nuclear matrix (64 +/- 9%). Also, in myeloblasts analyzed by immunofluorescence during mitosis, p48v-myb appeared to be dispersed through the cell like the lamina-nuclear matrix complex. Strong attachment to the nuclear matrix-lamina complex suggests that p48v-myb may be involved in DNA replication or transcription or both.

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