Clonal isolate of the simian sarcoma virus codes for a gag-related 65,000-dalton protein

VIROLOGY

114, 124-131 (1981)

Clonal Isolate of the Simian Sarcoma for a Gag-Related 65,000-Dalton EDWARD

Virus Codes Protein’

H.-JURGEN THIEL,*P~,~,~ THOMAS J. MATTHEWS,* M. BROUGHTON,*KENT J. WEINHOLD,* DAN1 P. BOLOGNESI,* BEUGt THOMAS GRAF,t AND HARTMUT

“Department of Surgery, D&e University Medical Center, Durham, North Carolina 27710,and ~Deutsc~ Krebsforschungszentrum, Institut fiir Virusfrschung, Heidelberg, West Germany Received March

18, 1981;

accepted May 20,

1981

An autologous antiserum against SSV nonproducer cells (SSV-NP cells) recognizes a 65,099-dalton protein (~65). This molecule does not appear to exhibit transformationspecific properties, but is serologically and biochemically very similar to the SSAV gagprecursor pr658=. Since p65 appears to be cleaved upon superinfection of SSV-NP cells with SSAV, it may provide a unique model to study gag-precursor cleavage of mammalian C-type viruses. INTRODUCTION

The genome of certain replication-defective transforming retroviruses has been shown to contain information coding for internal structural antigens (gag), which is probably derived from the respective helper viruses through recombination (Scolnick et aZ., 1973; Frankel and Fischinger, 1976; Donoghue et al., 1979). In the Moloney murine sarcoma virus (MoMSV) system, different isolates like mlMSV, m3-MSV, and 124-MSV encode gagrelated polypeptides of molecular weights between 60,000 and 70,000 (Oskarsson et cd., 1975, 1977, 1978; Robey et aZ., 1977; Philipson et aZ., 1978). The genomes of two other MO-MSV isolates, HTl-MSV and NP-MSV, contain a gag-region, but gagi This research was supported by Contract No. NC1 NO1 CP33308 of the Virus Cancer Program and U. S. Public Health Service Grant No. PO1 CA25863 from the National Cancer Institute. DPB is the recipient of an ACS Faculty Research Award, FRA141. * To whom reprint requests should be addressed. 3 On leave of absence from Max-Planck-Institut fur Virusforschung, Tiibingen, West Germany. ClPresent address: Bundesforschungsanstalt fiir Viruskrankheiten der Tiere, P.O. Box 1149, Paul-Ehrlich-Str. 28, 74 Ttibingen, West Germany. 0042-6822/81/130124-08$02.60/O Copyright All rights

0 1981 by Academic Press, In;. of reproduction in any form reserved.

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related polypeptides are not expressed in transformed cells (Huebner et al., 1966; Aaronson et al., 1972; Barbacid et al., 1976; Robey et cd., 1977). The situation is somewhat similar in the simian sarcoma virus (SSV) system, where Aaronson et al. (1975) described the differential expression of helper virus gagpolypeptides in cells transformed by clonal isolates of SSV. These studies suggest that transformation by MO-MSV and SSV can occur independently of the expression of gag-gene coded polypeptides. We recently undertook a detailed analysis of antigens in SSV-transformed cells with the intention of defining those entities which were associated with transformation by this virus (Thiei et al., 1981b, c). We describe here the identification of a 65,000-dalton molecule (~65) in SSV-NP cells, which is nearly indistinguishable from the SSAV gag-precursor (pr65g”g). Although this molecule does not appear to exhibit transformation-specific properties, it may provide a unique model to study gag-precursor cleavage of mammalian C-type viruses. MATERIALS

AND METHODS

Cells. SSV-transformed nonproducer NRK clones 9, 2-8, 11-2-3 (NRK-SSV-NP)

GAG-RELATED

PROTEIN

IN SSV NONPRODUCER

were obtained from Dr. S. A. Aaronson (Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland). Goat-SW-NP clones have been isolated in this laboratory (Thiel et al., 1981b). The SSV-NP clones from NRK cells (clone C2) and goat cells (clone 22) were isolated by infecting normal cells with various dilutions of SSV(SSAV) and resuspending the cells in methylcellulose. SSV-transformed colonies were then isolated with a drawn out Pasteur pipet and shown to be SSV nonproducer cells by the following criteria: (1) cell-free supernatants negative in a focus assay on NRK cells, (2) rescue of the SSV-genome after infection with SSAV, and, (3) lack of expression of SSAV gp70 (Thiel et al., 1971b). All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). Assay for focus formation. NRK cells (5 x 104) were plated in 24-well tissue culture trays in the presence of 2 pg/ml polybrene and allowed to settle overnight. The cells were infected by adding 0.1 ml of appropriately diluted virus. Virus was adsorbed for 60 min before the cultures were given maintenance medium. Foci were counted 7 days after infection. Superir$ection of nonproducer clone. The NRK-SSV-NP clone C2 was infected with SSAV in the presence of 2 pg/ml of polybrene. One week later the supernatants were tested for virus production by the NRK focus assay. Antisera. Preparation of serum against SSAV p30 has been described (Thiel et al., 1978). The rabbit serum against Rauscher murine leukemia virus (R-MuLV) p15 was kindly provided by Erwin Fleissner. Goat serum against autologous SSV(SSAV)-infected cells has been shown to react predominantly with gp70 and p15(E)/p12(E) (Deinhardt et al., 1978). Goat cells, nonproductively transformed by SSV, were used to prepare an autologous antiserum (Thiel et al., 1981b). The SSV-NP clone C2 has been shown to produce noninfectious particles (C2 particles), whose physical properties are very similar to infectious C-type viruses. In

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addition, the major structural component of these particles is p65 (unpublished). In order to prepare an antiserum against ~65, particles were recovered from supernatant fluids of the cell culture. After SDS-PAGE of purified particles, the gel was stained for 10 min and the 65,000 dalton bands cut out. The gel bands were homogenized in the presence of complete Freund adjuvant and injected four times subcutaneously into a rabbit. Radioactive labeling of cells and viruses. Radioactive labeling of cells and viruses with protein precursors generally followed previously described procedures (Deinhardt et al., 1978). Specific pulse-chase labeling procedures are indicated under Results. Immunopreci&ation and analysis bg polyacrylamide gel electrophoresis (SDSPAGE). Immunoprecipitates were formed as described earlier (Deinhardt et al., 1978), analyzed on slab gels (Laemmli, 1970), and processed for fluorography according to published procedures (Bonner and Laskey, 1974). Tmptic peptide analysis. The radioimmunoprecipitate from [3H]leucine-labeled (2 hr pulse) SSAV-infected cells was mixed with about 25 pg of nonradiolabeled C2 particles and electrophoresed on a preparative SDS-polyacrylamide gel. The [3H]leucine SSAV pr65gBgwas known to comigrate with the nonlabeled C2 p65 and this band was cut from the gel after visualization by staining. In a similar way the [‘4C]leucine-labeled C2 p65 was isolated. The [3H]SSAV pr65g”pand the [‘“C]C, p65 polyacrylamide gel bands were combined, 500 pg BSA added as carrier, and the polypeptides extracted from the gel slices with 60% formic acid for 4 hr at room temperature. Gel particles were removed by centrifugation and the sample was dried under a stream of nitrogen. The residue was dissolved in 900 ~10.05 M Tris, pH 8.0,300 ~1 DTT (12 mg/ml) was added, and the sample incubated at 37” for 30 min. About 600 ~1 iodoacetamide (40 mg/ ml) was added and incubation continued at 0” for 60 min. The polypeptides were precipitated by addition of 1.8 ml 50% TCA and centrifugation. The precipitate

126

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ET AL.

FIG. 1. (a) Immunoprecipitation analyses with SSV(SSAV). NRK-SSV(SSAV)-producing cells were labeled for 24 hr with [%I]leucine. The harvested virus (2 X lo5 cpm) was incubated with 2 ~1 of serum. SSV(SSAV) with (1) SSV-NP serum, (2) preimmune goat serum, (3) g-SSV serum, and (4) random pooled normal goat serum. Markers refer to SSAV proteins. (b) Immunoprecipitation analyses with SSAV-infected NRK cells. Cells were labeled for a 3 hr pulse with [SH]leucine, extracted, and 5 X lo6 cpm were incubated with 2 ~1 of serum. NRK-SSAV cells with (1) SSV-NP serum, (2) preimmune goat serum, (3) rabbit anti-SSAV p30 serum, and (4) normal rabbit serum. Markers refer to SSAV p16f9~ and SSAV ~30.

was rinsed with ether and dried under nitrogen. The protein was dissolved in 1 ml 0.1 M ammonium bicarbonate and digested for 24 hr at 37” with 25 pg trypsin. Fresh trypsin was added after the first 15 hr. The sample was lyophilized and the residue was dissolved in 100 ~1 8 M urea containing 5% 2-mercaptoethanol. The peptide fragments were analyzed by isoelectric focusing as described (Righett and Chillemi, 1978) except that a 0.6 X 18-cm tube gel was used rather than a slab gel and 0.01 M phosphoric acid and 0.01 M sodium hydroxide were used as anolyte and catholyte. After electrophoresis the gel was fractionated into 1 mm slices with a Gilson automatic gel fractionator and each slice was counted in Tritosol. About 75% of both the 8H and 14Cradiolabel applied to the gel was recovered. Peptide mapping b limited proteolysis in an SDS-pol~acrglamide gel. The [%I]leutine-labeled SSAV pr65Wg and [3H]leutine-labeled C2 ~65 were obtained as gel slices from preparative gels as described in the tryptic peptide method (above). The gel slices were placed onto a 15% polyacrylamide gel (acrylamide to bisacrylamide, 4O:l) containing a 4.5-cm stacking

gel. The slices were overlayed with 5 pg Staphylococcus aureus V8 protease and electrophoresis was as described (Cleveland et al, 1977). RESULTS

Antiserum against SSV-NP Cells Recognizes SSA V gag-Molecules Results presented in a previous manuscript showed that an autologous antiserum against SSV-NP cells contained reactivity against purified SSAV p30 as measured by a radioimmunoassay (Thiel et al., 1981b). In order to analyze the reactivity of the SSV-NP serum against other SSAV structural proteins, metabolically labeled virus SSV(SSAV) was used for immunoprecipitation (Fig. la). Under these conditions, the major core protein ~30, and the other three gag-gene coded proteins ~15, ~12, and p10 are precipitated. The serum shows no reactivity with the envgene products gp70 and p15(E)/p12(E), which are recognized by an autologous goat serum (g-SSV-S) against SSV(SSAV)infected cells (Lane 3). Migration characteristics of SSAV p12 were established through immunoprecipitation of [“PI-

GAG-RELATED

PROTEIN

IN SSV NONPRODUCER

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127

molecule precipitated by SSV-NP serum from the goat SSV-NP cells used for imOP70 munization (clone 22), is a 65,000-dalton molecule (~65). A molecule with the same migration characteristics is also found in P30 an NRK SSV-NP clone (C2) after immunoprecipitation with the SSV-NP serum (Fig. 3, Lane 2). In both cases cells were pulse labeled for 7 hr with [3H]leucine. Lower molecular weight proteins could not be detected, suggesting that p65 is not cleaved during the labeling period. Pulsechase experiments were then undertaken to follow processing of p65 more closely. As can be seen in Fig. 3, the p65 band FIG. 2. Immunoprecipitation analyses with goat shows no consistent change during a 1-12 SSV-NP cells (Clone 22). Cells were labeled for a 7 hr chase period and there is no precipihr pulse with rH]leucine, extracted, and 3 X 10’ cpm were incubated with 2 ~1 of serum. Molecular weight tation of cleaved polypeptides, indicating markers refer to (1) [‘Hlleucine-labeled Friend leuthat p65 is not processed in NRK SSVkemia virus (FLV). Goat SSV-NP cells with (2) transformed nonproducer cells. Some preimmune goat serum, and (3) SSV-NP serum. bands of lower molecular weight than p65 appear to be specifically precipitated by orthophosphate-labeled virus, a procedure the SSV-NP serum. While these have not which results in specific labeling of this been further characterized, their intensity component (not shown). Identification of does not consistently change during the SSAV p15 was accomplished by including chase period indicating that there is no an anti-Rauscher MuLV p15 serum, which precursor-product relationship between precipitates SSAV p15 through interspep65 and these proteins. When goat SSVties reactivity (not shown, Thiel et aZ., NP cells were used for pulse-chase exper1981a). iments there was also no indication of-p65 We also examined the reactivity of this cleavage (not shown). serum with cell extracts of SSAV-infected NRK cells which were pulse labeled for 3 12 345 67 89lOll hr with [3H]leucine. Under these conditions, the SSV-NP serum precipitates a 65,000-dalton molecule (Fig. lb), which probably represents the gag-precursor pr65gw; the same molecule is also precipitated by anti-SSAV p30 serum (Lane 3). There is no indication that the serum reacts with the env-precursor pr90”“‘. These data indicate that the SSV-NP serum precipitates the SSAV gag-precursor pr65g8gas well as the four processed FIG. 3. Pulse chase studies with NRK SSV-NP cells gag-proteins. (Clone C2). Cells were labeled for a 7 hr pulse with Precipitation

of ~65 from SSV-NP Cells

Because antibodies against all four SSAV gag-protein determinants were produced after autologous immunization with SSV-NP cells, we wanted to determine if the processed molecules are present in SSV-NP cells. Figure 2 shows that the only

rH]leucine, chased for varying lengths of time, extracted, and incubated with 1 ~1 of serum. Molecular weight markers refer to (1) [‘Hlleucine-labeled FLV. NRK SSV-NP cells after a 7 hr pulse with (2) SSVNP serum, (3) preimmune goat serum; after 1 hr chase with (4) SSV-NP serum, (5) preimmune serum; after 3 hr chase with (6) SSV-NP serum, (7) preimmune serum; after 6 hr chase with (8) SSV-NP serum, (9) preimmune serum; after 12 hr chase with (10) SSV-NP serum, and (11) preimmune serum.

128

THIEL

Relatednessof p65 to SSAVStructural Proteins Because of the reactivity of the SSV-NP serum with the SSAV-gag gene products and the similar molecular weights of p65 and pr65eag,p65 was analyzed directly for the presence of SSAV gag-determinants. For this, monospecific sera, including an anti-SSAV p30 serum and an antiRauscher MuLV p15 serum, were reacted against SSV-NP cell extracts (Fig. 4). That both sera clearly precipitate ~65 indicates that this molecule possesses p15 as well as p30 determinants. The anti-p30 serum precipitated, in addition to ~65, a lower molecular weight protein. This molecule migrated slightly faster than SSAV p30 and has been termed ~27. P27 was not precipitated by the SSV-NP serum (not shown) and is, therefore, considered to be unrelated to ~65. Because there are no monospecific sera available against SSAV p12 and ~10, the presence of these determinants on p65 could not be investigated by this approach. We therefore raised an antiserum by immunizing a rabbit with p65 obtained directly from SDS-PAGE (see Methods). Figure 5 shows that this serum precipitates p65 from SSV nonproducer cells as well as the gag proteins ~15, ~12, ~30, and p10 from labeled SSV(SSAV) virions (Fig. 5). In addition the anti-p65 serum was shown to precipitate iodinated SSAV p12 I234

FIG. 4. Presence of SSAV gag determinants on ~65. Cells were labeled for a 4 hr pulse with [sH]leucine, extracted, and 5 X 106 cpm incubated with 2 ~1 of serum. NRK SSV-NP cells with (1) SSV-NP serum, (2) preimmune goat serum, (3) rabbit anti-SSAV p30 serum, and (4) rabbit serum anti-R-MuLV ~15.

ET AL. 1234

FIG. 5. Reactivity of rabbit anti-p65 antiserum. NRK SSV-NP cells were labeled for a 4 hr pulse with [3H]leucine, extracted, and 5 X lo6 cpm incubated with 2 ~1 of serum. NRK SSV-NP cells with (1) rabbit serum antip65, (2) preimmune rabbit serum. NRK SSV(SSAV)-producing cells were labeled for 24 hr with [3H]leucine. The harvested virus (2 X ld cpm) was incubated with 2 pl of serum. SSV(SSAV) with (3) rabbit serum anti-p65, and (4) preimmune rabbit serum. Markers refer to SSV p65 and SSAV structural proteins.

in a direct radioimmunoassay (M. Barbacid, personal communication). It thus appears that p65 shares determinants with these four polypeptides.

Comparison of p65 with the SSAVgag-Precursor To further analyze the serological relationship of p65 and gag-molecules, the SSAV gag-precursor was compared with p65 by peptide mapping. For this, SSV-NP cells and NRK-SSAV cells were pulse labeled for 2 hr with rH]leucine. After immunoprecipitation of both molecules with an anti-SSAV p30 serum the samples were electrophoresed on a preparative SDS gel, the respective 65,000-dalton bands cut out, and peptide analyses of these molecules were performed as described by Cleveland et al. (1977). Lanes 1 and 2 in Fig. 6a demonstrate that p65 and the SSAV gag-precursor migrate the same distance into this 15% polyacrylamide SDS gel. This can also be observed, when lower polyacrylamide concentrations (7.5-12s) are used (not shown). After limited digestion of both molecules with Staph A protease (lanes 3-4), the peptide maps show a high

GAG-RELATED

PROTEIN IN SSV NONPRODUCER CELLS

FRACTION

123

NUMBER

FIG. 6. (a) Comparison of SSAV pr6F and SSV-NP p65 by limited proteolysis in an SDSpolyacrylamide gel. The [*H]leucine-labeled SSAV pr65m (lanes 1 and 3) and SSV-NP p65 (lanes 2 and 4) containing bands were isolated from a preparative SDS-polyacrylamide gel and placed on the 15% analytical SDS-polyacrylamide gel. Samples were overlayed with Staphylococcus aureus V8 protease (lanes 3 and 4) and electrophoresed as described under Materials and Methods. (b) Comparison of [sH]leucine-labeled SSAV pr6F (. . . . ) and [‘Clleucine-labeled SSV-NP p65 (-) by isoelectric focusing of their tryptic fragments. The polypeptide chains were isolated, digested with trypsin, and the tryptic fragments electrophoresed as described under Materials and Methods. About 70,666 cpm of the *H fragments and 12,606cpm of the “C fragments were applied to the gel.

degree of homology, but there is one clear difference with this proteolytic enzyme (arrow). Labeling of the two molecules with [?S]cysteine also results in one difference by peptide mapping, while [%]methionine-labeled p65 and pr65gBB appear identical after limited digestion (not shown). In addition, tryptic peptide maps were carried out by labeling p65 with [i4C]leucine and pr65g*g with rH]leucine. After immunoprecipitation, both molecules were digested and electrophoresed on the same cylindrical gel. Figure 6b shows that tryptic maps also result in only one clear difference between the two molecules (arrow). This indicates that in spite of identical migration characteristics on SDS gels as well as their close serological relationship, p65 and the SSAV gag-precursor can be distinguished by peptide mapping.

ducer cell line with a helper virus. We used SSAV for infection of the SSV-NP clone and carried out immunoprecipitation analyses of labeled cell extracts after a 1.5 hr pulse as well as a 1.5 hr pulse followed by 14 hr chase (Fig. 7). The SSV-NP serum recognizes a 65,000-dalton molecule as well as p30 from pulse-labeled cells, analogous to the results obtained from SSAV-infected NRK cells (see Fig. lb). After the chase period, the 65,000-dalton molecule is not detectable, while SSAV p30 is clearly precipitated. A similar result was obtained, when NRK-SSAV-infected cells were reacted using the above mentioned labeling conditions (not shown). This suggests that not only pr65g88,but also ~65, is processed in these cells after superinfection with SSAV.

Behavior of p65 after Superinfection

Analysis of an autologous antiserum against SSV .nonnroducer cells revealed & reactivity against the SSAV gag-gene coded proteins ~15, ~12, ~30, and ~10. The SSV-NP clone, which was used for immunization, did not contain these pro-

The high degree of similarity between p65 and pr65= suggested the possibility that p65 might be processed after superinfection of the respective SSV nonpro-

DISCUSSION

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THIEL

ET AL.

but no p10 (Oskarsson et aZ.,1978). In contrast, SSV p65 not only possesses determinants of all four gag-proteins, but is nearly indistinguishable from the SSAV gag-precursor by peptide mapping. The minor differences observed might be due to post-translational modification, One distinction between pr65g”p and ~65 is that the former is processed while the latter is not, at least in SSV-NP cells. We, therefore, attempted to cleave p65 by superinfecting the respective SSV nonproducer clone with SSAV. The results indicate that p65 is processed under these conditions like SSAV pr65gag,at least with regard to FIG. ‘7. Immunoprecipitation from NRK SSV-NP SSAV ~30. Since p65 is so similar to the cells C2 after superinfection with SSAV. Cells were gag-precursor, the data suggest that the labeled with [sH]leucine either for a 1.5 hr pulse presence of the helper virus is required to (lanes 2 and 3) or a 1.5 hr pulse followed by a 14 hr chase (lanes 4 and 5). Cell extracts (5 X lo6 cpm) were cleave ~65. incubated with 1 pl of serum. Molecular weight markIn the avian C-type virus system, the ers refer to (1) [aI-I]leucine-labeled FLV. C2(SSAV) gag-precursor pr76 is cleaved in vitro into after a 1.5 hr pulse with (2) preimmune goat serum, internal structural proteins by the C-ter(3) SSV-NP serum; CZ(SSAV) after a 1.5 hr pulse 14 minal viral protein, p15 (Von der Helm, hr chase with (4) SSV-NP serum, (5) preimmune goat 1977). Yoshinaka and Luftig (1977) have serum. shown that murine leukemia viruses contain a proteolytic factor, which cleaves the cessed molecules, but instead expresses a gag-precursor and is different from the 65,000-dalton molecule (p65), which mi- four gag polypeptides. Our data in the grates like the SSAV gag-precursor ~165~~ SSV-system indicate that the intracellular on SDS gels. Further analysis showed that gag-precursor cleavage occurs only when p65 contains determinants of all four gag- the helper virus is present. We do not proteins and that it is not detectably pro- know if the helper virus activates a celcessed during a 12 hr chase period. lular protease or if it actually codes for a proteolytic enzyme. Alternatively, producUsing competition radioimmunoassays for SSAV gag-proteins, Aaronson et al. tion of the helper virus might be necessary (1975) described clonal isolates of SSV for optimal processing of the gag-precurwith differential expression of helper vi- sor by cellular enzymes. In order to make these conclusions more rus structural polypeptides. We have assayed these NRK clones by radioimmudefinitive, however, it must be demonnoprecipitation. As expected, p65 was strated that p65 is still synthesized after found only in the SSV-NP clone (11-2-3) superinfection with SSAV. If p65 is exwhich tested positive for all SSAV gag pressed, peptide mapping of the 65,000proteins (unpublished). dalton region should reveal p65 as well as Detection of helper virus polypeptides pr6588Bspecific peptides and preliminary has been described in Moloney sarcoma results demonstrate that this is indeed the virus-transformed cells (Oskarsson et d., case. It thus appears that this system can 19’75, 1977, 1978; Robey et al., 1977; Philbe utilized to study the mechanism as well ipson et al., 1978). As in the SSV system, as the specificity of the gag-precursor transformation by MoSV appears to occur cleavage of mammalian C-type viruses. independently of the expression of helper virus polypeptides. The ml-Moloney-MSV ACKNOWLEDGMENTS codes for a 60,000-dalton molecule, termed pr60 or pP6pag, which according to peptide The authors thank J. N. Ihle and E. Wecker for mapping, contains ~15, ~12, part of ~30, stimulating discussions; S. A. Aaronson and M. Bar-

GAG-RELATED

PROTEIN IN SSV NONPRODUCER CELLS

bacid for their valuable contributions to this manuscript; M. Viana and J. Mills for excellent assistance, as well as D. Wilson for typing the manuscript. REFERENCES AARONSON,S. A., BASSIN, R. H., and WEAVER, C. (1972). Comparison of murine sarcoma viruses in non-producer and SL- transformed cells. J. ViroL 9.701-794. AARONSO& S. A., STEPHENSON,J. R., HINO, S., and TRONICK, S. R. (1975). Differential expression of helper viral structural polypeptides in cells transformed by clonal isolates of woolly monkey sarcoma virus. J. ViroL 16, 111’7-1123. BARBACID, M., STEPHENSON,J. R., and AARONSON, S. A. (1976). The gag gene of mammalian type-C RNA tumour viruses. Nature (hdon) 262, 554559. BANNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. B&hem. 46,83-88. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. BioL Chem. 252. 1102-1106. DEI~‘HARDT. F., BERGHOU, C., HUNSMANN, G., SCHNEIDER,J., THIEL, H.-J., BEUG,H., and SCZH~~ER. W. (1978). Studies of simian sarcoma and simian sarcoma-associated virus. I. Analysis of viral structural proteins, and preparation and characterization of antiserum specific for viral envelope components. 2. Nuturforsch. 33~. 969-980. DONOGHUE.D. J., SHARP, P. A., and WEINBERG, R. A. (1979). Comparative study of different isolates of murine sarcoma virus. J. ViroL 32, 10151027. FRANKEL, A. E., and FISCHINGER,P. J. (1976). Nucieotide sequences in mouse DNA and RNA specific for Moloney sarcoma virus. Proc. Nat. Acad. Sci. USA 73.3705-3709. HUEBNER,R. J., HARTLEY, J. W., ROWE,W. P., LANE, W. T., and CAPPS, W. I. (1966). Rescue of the defective genome of Moloney sarcoma virus from a non-infectious hamster tumor and the production of pseudotype sarcoma viruses with various murine leukemia viruses. Proc. Nat. Acad. Sci. USA 56, 1164-1169. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (kndon) 227.680-685. OSKARSSOK,M. K., ROBEY,W. G., HARRIS, C. L., FISCHINGER. P. J., HAAPALA, D. K., and VANDE WOUDE,G. F. (1975). A p60 polypeptide in the feline leukemia virus pseudotype of Moloney sarcoma virus with murine leukemia virus p30 antigenic

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determinants. Proc. Nat. Acd Sci. USA 72,239& 2384. OSKARSSON,M. K., LONG, C. W., ROBEY, W. G., SCHERER.M. A., and VANDE WOUNDE,G. F. (1977). Phosphorylation and nucleic acid binding properties of ml Moloney murine sarcoma virus-specific pPv. J. ViroL 23, 196-m. OSKARSSOK,M. K., ELDER, J. H., GAUTSCH,J. W., LERNER. R. A., and VANDE WOUDE. G. F. (1978). Chemical determination of the ml Moloney sarcoma virus pPww gene order. Pra. Nat. Acad S& USA 75.4694-4698. PHILIPSON,L., ANDERSSON,P., OLSHEVSKY,U., WEINBERG, R., BALTIMORE, D., and GESTELAND, R. (1978). Translation of MuLV and MSV RNAs in nuclease-treated reticulocyte extracts: Enhancement of the gag-pol polypeptide with yeast suppressor tRNA. CeU 13, 189-199. RIGHEIT, P. G., and CHILLEMI, F. (1978). Isoelectric focusing of peptides. J. Chromatogr. 157.243251. ROBEY, W. G., OSKARSSON,M. K., VANDE WOUDE, G. F., NASO, R. B., ARLINGHAUS,R. B.. HAAPALA, D. K., and FISCHINGER,P. J. (1977). Cells transformed by certain strains of Moloney sarcoma virus contain murine p60. CeU 10.79-89. SCOLNICK, E. M., RAND& E., WILLIAMS, D., and PARKS, W. P. (1973). Studies on the nucleic acid sequences of Kirsten sarcoma virus: a model for formation of a mammalian RNA-containing sarcoma virus. J. ViroL 12, 458-463. THIEL, H.-J., BEUG, H., GRAF, T., SCHWARZ,H., SCH;~FER,W., BERGHOLZ,C., and DEINHARDT, F. (1978). Studies of simian sarcoma and simian sarcoma-associated virus. II. Isolation of the major viral glycoprotein, properties of this component and its specific antiserum. Firoh 96,360-365. THIEL. H.-J., BROUGHTON,E. M., MATTHEWS.T. J., SCHAFER,W., and BOUK~NESI,D. P. (1981a). Interspecies reactivity of type C and D retrovirus p15E and p15C proteins. Virdogy, 111.270274. THIEL, H.-J., ICLEHART, J. D., MA~EWS, T. J., and BROUGHTON,E. M. (1981b). Preparation of autologous antiserum against SSV non-producer cells and its partial characterization. Virdogy, 112,634til. THIEL, H.-J., MATI-HEWS, T. J., BROUGHTON, E. M., BUTCHKO, A. W., and BOLOCNESI,D. P. (1981c). Detection of a transformation specific glycopeptide in SSV-infected cells. Virology, 112, 642-650. VONDERHEM, K. (1977). Cleavage of Rous sarcoma viral polypeptide precursor into internal structural proteins in vitro involves viral protein p15. Proc. Nat. Acad Sci. USA 74,911-915. YOSHINAKA,Y., and LUFTIG, R. B. (1977). Properties of a ~‘70 proteolytic factor of murine leukemia viruses. Cell 12. 799-719.