IDENTIFICATION OF TRYPTIC PEPTIDES UNIQUE TO A I10,000-MOLECULA R WEIGHT POLYPROTEIN ENCODED BY THE T-8 ISOLATE OF MURINE LEUKEMIA VIRUS

Several type C RNA-transforming viruses have been shown to contain acquired cellular sequences within preexisting viral genes (I-3). In many instances expression of proteins encoded by such sequences occurs by inphase read through of translation initiated at the 5' terminus of the viral genome. Resulting polyprotein translational products contain type C viral gag gene amino terminal structural components covalently linked to acquired sequence-encoded components with probable transform- ing function (4-10). The nonstructural components of polyproteins encoded by several such virus isolates have been shown to resemble the well-characterized avian sarcoma virus-encoded transforming protein (11-13), pp60 src, in that they are highly phospho- rylated (14) and exhibit associated protein kinase activity (14, 15) with specificity for tyrosine acceptor sites (15-18). Transformation by viruses of this group is associated with elevated levels of total cellular phosphotyrosine (17, 18) and a reduction of epidermal growth factor (EGF) a binding capacity (16, 19). Staa( et al. (20) have described the isolation of a weakly-transforming RNA type C virus from a thymoma suspension culture developed from a spontaneously lympho- matosis AKR/J mouse. This virus, designated T-8, is replication-defective (20), transforms mink cells in vitro (20), and encodes, as its major translational product, a highly phosphorylated 110,000 mol wt polyprotein containing AKR-murine leukemia virus (MuLV) amino terminal gag gene components, p 15 and p 12 (21). The functional significance and role, if any, of this protein in transformation is, as yet, unknown. In the present study, we have analyzed T-8 P 110 for possible acquired sequence encoded component(s) and have examined levels of tyrosine-specific protein kinase activity and epidermal growth factor EGF binding by mink cell lines nonproductively infected by T-8. * Supported under Public Health Service contract N01-CO-75380 from the National Cancer Institute, Bethesda, Md. i Abbreviations used in this paper: DME, Dulbeceo's modification of Eagle's medium; EGF, epidermal growth factor; MCF, mink cell focus forming; MuLV, murine leukemia virus; PBSTDS, 10 mM sodium phosphate, pH 7.2, 0.9% NaCI, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate; SDS, sodium dodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TPCK, tolyl- sulfonyl phenylalanyl chloromethyl ketone.