Vol. 23, No. 1 Printed in U.S.A.

JOURNAL OF VIROLOGY, July 1977, p. 74-79 Copyright © 1977 American Society for Microbiology

Type C Viral gag Gene Expression in Chicken Embryo Fibroblasts and Avian Sarcoma Virus-Transformed Mammalian Cells FRED H. REYNOLDS, JR., CHARLOTTE A. HANSON, STUART A. AARONSON, AND JOHN R. STEPHENSON* Viral Oncology Program, NCI Frederick Cancer Research Center, Frederick, Maryland 21701,* and Laboratory of RNA Tumor Viruses, National Cancer Institute, Bethesda, Maryland 20014

Received for publication 16 February 1977

Sensitive radioimmunoassays were developed for avian type C viral gag genecoded proteins. These assays were used to examine the restriction to virus production by avian embryo cells and mammalian cells transformed by avian sarcoma viruses. The results indicate that although a high-molecular-weight primary translational product of the gag gene is expressed, its cleavage and processing are incomplete. Furthermore, analysis of intermediate cleavage products provided information regarding the order of sequences coding for the individual viral proteins within the avian type C viral gag gene.

Studies of avian RNA tumor viruses have led to the identification of four distinct regions of the viral genome. These include genes coding for the RNA-dependent DNA polymerase, the envelope glycoprotein, and several lower-molecular-weight proteins synthesized in the form of a common precursor (7, 28). These regions of the viral genome have been designated poi, env, and gag, respectively (2). A fourth viral gene, src, has been implicated in malignant transformation (8, 16, 19). The relative positions of the known structural genes from the 5' to 3' end of the avian sarcoma virus genome have tentatively been established as gag-polenv-src (15, 30 to 32). Several approaches have been used to map regions of the gag gene of type C RNA viruses coding for individual viral structural proteins. One technique that has been applied to studies of avian type C viruses has been the use of pulse-labeling experiments with pactamycintreated, virus-infected embryo cultures (9, 28, 29). The results of these studies have provided suggestive evidence for the presence of a 19,000molecular-weight protein (p19) at the aminoterminal end of the avian type C viral gag gene product and a 15,000-molecular-weight protein (p15) at its carboxy terminus. An approach used in mapping the mammalian type C viral gag gene has involved the application of competition immunoassays, specific for individual viral proteins, to the analysis of the components of the gag gene precursor protein and its intermediate cleavage products (3). 74

The possibility that the gag gene of avian type C viruses might be mapped by an approach analogous to that used for the mammalian type C viral genome was suggested by the demonstration that certain chicken embryo cells (5, 6, 25), as well as several avian tumor virus-transformed mammalian cell lines (9, 22), express gag gene translational products in the absence of infectious virus production. In the present study, such cells were analyzed by competition immunoassay for the presence of gag gene precursor polypeptides and their intermediate cleavage products in an attempt to order genetic sequences coding for individual viral proteins within the avian type C viral gag gene. MATERIALS AND METHODS Viruses. Avian myeloblastosis virus (AMV) in chicken plasma, supplied by J. Beard, and the Prague strain of Rous sarcoma virus (Prague RSV),

from University Laboratories, were obtained through the courtesy of J. Gruber, Office of Resources and Logistics, National Cancer Institute. Chicken embryos and avian sarcoma virus-transformed cell lines. Leukosis-free chicken embryos were obtained from SPAFAS Inc., Norwich, Conn. Several RAV-0-positive chicken embryos (line 100) were a gift of L. B. Crittenden, U.S. Department of Agriculture Regional Poultry Research Laboratory, East Lansing, Mich. A clonal Schmidt-Ruppin RSVtransformed rat kidney cell line, SR-NRK, and the XC cell line, originally derived from a Wistar rat tumor induced with Prague RSV, have been described (23, 27). Preparation of cell extracts. Embryo cell extracts were prepared by homogenization of minced 12-day

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TYPE C VIRAL gag GENE EXPRESSION

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whole embryos for 30 s in an equal volume of 0.01 M the same buffer. The column was washed with the Tris-hydrochloride (pH 7.8), 1 mM EDTA, 0.01 M same buffer, and p15 eluted in the wash fraction NaCl, 1 mM dithiothreitol, and 10% glycerol. Em- while the major contaminants adhered to the colbryo cell homogenates and tissue culture fibroblasts umn. resuspended in the same buffer were subjected to Radioimmunoassays. Viral proteins were labeled sonic treatment for 30 s (Biosonic II sonic oscillator) with 125I (14 mCi/mol; Amersham/Searle, Arlington and centrifuged at 20,000 rpm for 30 min in a Beck- Heights, Ill.) by the chloramine T method (12). Douman type 30 rotor. Post-microsomal supernatants ble-antibody competition radioimmunoassays were were tested for protein by the method of Lowry et al. performed as described previously (23). Serial two(18), divided into equal portions, and stored at fold dilutions of purified proteins, detergent-disrupted virus, or tissue homogenates were tested for -700C. Purification of viral proteins. Viral proteins were the ability to compete with 125I-labeled viral proteins purified from density gradient-purified AMV. Virus for binding limiting amounts of porcine antisera was disrupted in 0.1 M Tris-hydrochloride (pH 9.0) prepared against AMV proteins. Reaction mixtures buffer containing 1.0% Triton X-100, sonically (0.2 ml) contained 0.01 M Tris-hydrochloride (pH treated for 30 s, and centrifuged at 20,000 rpm in a 7.8), 0.4% Triton X-100, 0.001 M EDTA, 1% bovine Beckman type 30 rotor for 30 min. The process was serum albumin, and either 0.05 M NaCl (p27 and repeated, and the pooled supernatants were dialyzed p12 immunoassays) or 0.3 M NaCl (p19 and p15 overnight against 200 volumes of BET buffer [0.01 M immunoassays). Unlabeled antigen and antisera N,N-bis- (2-hydroxyethyl)-2-amino-ethanesulfonic were incubated at 37°C for 1 h. 125I-labeled antigen acid (pH 6.5), 0.001 M EDTA, and 0.5% Triton X- was added with incubation at 37°C for 3 h and then 100]. Solubilized proteins were applied to a phospho- at 4°C for 18 h. Goat antiserum against porcine cellulose column (1.5 by 5 cm; Whatman P11, H. immunoglobulin G (0.025 ml) was then added to Reeve Angel and Co., Clifton, N.J.) equilibrated each reaction. After incubation for 1 h at 37°C and with BET. The column was washed with 50 ml of an additional 6 h at 4°C, the precipitate, obtained by BET, and 2-ml fractions of a 0.0 to 0.5 M linear KCl centrifugation at 2,500 x g for 15 min, was counted gradient were collected. Individual viral structural in a gamma scintillation counter. All antisera were proteins were localized by sodium dodecyl sulfate- generously provided by R. Wilsnack through the polyacrylamide gel electrophoresis (SDS-PAGE) or Office of Resources and Logistics, National Cancer competition radioimmunoassay as previously de- Institute. Agarose gel column chromatography. Cell exscribed (4, 5, 13, 17, 20, 22, 26). Unbound proteins in the wash containing pre- tracts were analyzed by agarose gel filtration in the dominantly p12 were lyophilized, resuspended in 0.2 presence of 6 M GuHCl according to previously deml of 0.05 M Tris-hydrochloride buffer (pH 8.5) con- scribed procedures (10). Briefly, around 25 mg of cell taining 0.01 M dithiothreitol, 0.002 M EDTA, and extract was lyophilized, resuspended in 0.2 ml of 8 M guanidine hydrochloride (GuHCl), applied to 0.05 M Tris-hydrochloride (pH 8.5), 0.01 M dithioan agarose A-0.5 (100 to 200 mesh) (Bio-Rad Prod- threitol, 0.002 M EDTA, and 8 M GuHCl, and apucts, Richmond, Calif.) column (1.5 by 90 cm), and plied to an agarose A-5 column (1.5 by 90 cm) in the subjected to gel filtration in the presence of sodium presence of sodium phosphate (pH 6.5)-6 M GuHClphosphate buffer (pH 6.5) containing 6 M GuHCl 0.01 M dithiothreitol buffer. Fractions (0.5 ml) were and 0.01 M dithiothreitol. Fractions eluting at the dialyzed for 18 h against 0.01 M Tris-hydrochloride same molecular-weight regions as l25I-labeled AMV buffer (pH 7.8) containing 0.1% Triton X-100 and p12 marker were dialyzed against Tris-hydrochlo- tested at serial twofold dilutions in competition imride buffer (pH 7.5) containing 0.01 M NaCl and munoassays for individual viral proteins. 0.1% Triton X-100, divided into equal portions, and RESULTS stored under liquid nitrogen. Proteins eluting from phosphocellulose between Molecular-size analysis of type C viral anti0.01 and 0.05 M KCl were concentrated with a hol- gens expressed in RAV-0-positive chicken emlow fiber device (Bio-Rad Products) and subjected to bryo cells. The four avian tumor virus strucfurther purification by gel filtration on an Ultrogel tural proteins isolated in the present study AcA 54 column (1.5 by 90 cm; LKB Products, Rockville, Md.) in 0.01 M Tris-hydrochloride buffer (pH were 125I-labeled by the chloramine T procedure 7.5) containing 0.1 M NaCl and 0.1% Triton X-100. as described in Materials and Methods. Each Fractions co-chromatographing with an 125I-labeled was shown by SDS-PAGE molecular-size analAMV p27 marker were divided into equal portions ysis to exhibit a high degree of radiochemical and stored under liquid nitrogen. Viral p19 eluting purity. Further, all four labeled antigens could from the phosphocellulose column between 0.1 and be over 90% precipitated by antibody to AMV, 0.2 M KC1 was further purified by agarose gel fil- but not by control sera prepared against type C tration in the presence of 6 M GuHCl as described viruses of mammalian origin. Competition imabove for p12. Viral p15, eluting from phosphocellu- munoassays developed by use of these 125I-lalose between 0.2 and 0.4 M KCl, was dialyzed inagainst 0.01 M Tris-hydrochloride buffer (pH 8.5) beled proteins were highly specific forIneach initial containing 0.001 M EDTA and 0.1% Triton X-100 dividual protein (data not shown). and applied to a DEAE-cellulose column (1.5 by 5 studies, embryo cells of line 100 chickens, cm; Whatman DE52) previously equilibrated with known to be constitutively positive for RAV-0

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TABLE 1. Type C viral antigen expression in chicken embryos and avian tumor virus-transformed mammalian cellsa Cells tested

Level of viral protein (ng of antigen/mg of cell extract) p15 p27 p19 p12

RAV-0-positive chicken embryos 6091 1,230 6092 1,650 995 6093 SPAFAS embryo cells 307 7320 40 7321 250 7322 490 7324 Mammalian fibroblast cultures 85 XC 65 SR-NRK NRK