The gag-mos hybrid protein of ts110 moloney murine sarcoma virus: variation of gene expression with temperature

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The gag-mos Hybrid Protein of tsll0 Moloney Murine Sarcoma Virus: Variation of Gene Expression with Temperature GARY E. GALLICK,* RICHARD HAMELIN,? STEVEN MAXWELL,t DEBORAH DUYKA,? AND RALPH B. ARLINGHAUSTl *Department

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Tumor Biology, Section of Virology, University of Texas, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030, and jThe Scripps Clinic and Research Foundation, La Jolla, Calzfornia 92097 Received June 15, 1984 accepted August 22, 1984

A NRK cell clone (6m2 cells) infected with tsll0 Moloney murine sarcoma virus (MuSV) produce a gag-mos protein, P&Y-“, and a truncated gag protein of M, 58,000d termed P58@‘@. The gag-mos protein is produced from a 3.5-kb mRNA whereas the gag protein is made from a 4.0-kb mRNA. It has been proposed that the 3.5-kb RNA is produced from the 4.0-kb RNA by a splicing mechanism (R. P. Junghans, E. C. Murphy, Jr., and R. B. Arlinghaus (1982) J. MoL BioL 161, 229-255). The results presented here provide further support for this model. The expression of the 3.5-kb RNA and the gag7120s protein increased as the temperature at which 6m2 cells were maintained was lowered from 39 to 28”. This increase coincided with a decrease in both the 4.0-kb RNA and its product P581n’“. The optimum temperature for syntheses of both the gag-mos mRNA and its protein was found to be 28”. Consistent with the increase in the level of the gag-mos protein is the increase in the protein kinase activity associated with P85U”“,,,‘I and the degree of morphological transformation of 6m2 cells. Thus, the level of P85”““~‘“‘” within 6m2 cells is directly proportional to the degree of cell transformation and the amount of the kinase activity associated with the gag-mos protein, providing convincing evidence that P85y’“-“‘I” plays a direct role in the neoplastic transformation of these cells. 0 1984 Academic Press. Inc. INTRODUCTION

mutant which at the permissive temperature produces easily detectable levels of Several types of retroviruses that have gag-mos hybrid protein (Stanker et cd, acquired sequences of the cellular mos 1983a). The relatively high levels of transgene have been identified. In prototype forming protein and ability to induce viruses such as Moloney murine sarcoma transformation by temperature shift make virus strain 124 (Ball et ok, 1973), the mos tsllO-infected cells important for studying gene has recombined with the leukemia transformation by v-mos. This variant virus in the env region. As a result, these strain has multiple defects which affect strains express an env-mos hybrid protein, phenotype. One defect termed ~37~. However, this product is the transformed produced in trace amounts (Papkoff et CLL, affects the stability of the 85,000-Da gagmos protein (Stanker et al, 1983b). A 1982), making the study of transformation second defect involves formation of the induced by p3T”” very difficult. A mutation mRNA for this protein. The RNA of the in MuSV 124, causing an internal deletion and in the genome (Junghans et cd, 1982), is tsll0 virus has been characterized found to contain two species with approxresponsible for a another type of Moloney imate sizes of 4.0 and 3.5 kb (Horn et ak, murine sarcoma virus. The variant virus, 1981). Both RNAs contain a central deletermed tsll0, is a temperature-sensitive tion which results in the fusion of the gag gene and wws gene segments (Jung’ Author to whom requests for reprints should be hans et al, 1982). The 4.0-kb RNA codes addressed. for a 58,000-Da truncated gag gene pro0042-6822/84 Copyright All righta

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duct whereas the 3.5-kb RNA encodes the 85,000-Da gag-~QS protein termed P858”6-“*.By use of the Sl n&ease technique, we have recently demonstrated that the 3.5-kb RNA is not detectable at the restrictive temperature, however, this RNA appears after shift to the permissive temperature (Nash et aL, 1984). Thus, the formation of the 3.5-kb RNA encoding PWW-“” appears to be temperature sensitive. This result agrees with our previous work which demonstrated that P58w is made and is stable at both the permissive temperature (33”) and nonpermissive temperature (39’), whereas P858”Q-“” is made at 33’, but is undetectable at 39” (Horn et aL, 1981). The experiments summarized above led us to propose that P85BWW expression results from a temperature-sensitive splicing event in which the 4.0-kb RNA loses internal sequences, resulting in 3.5kb message which contains gag and mos sequences fused in an open reading frame (Junghans et &, 1982). Several predictions arise from this model. If the 3.5-kb message were formed from the 4.0-kb message, conditions which increase levels of this smaller message should decrease levels of the larger message. Concomitantly, conditions which increase levels of P8p-“” should decrease levels of P588”g. Furthermore, functions specifically associated with P85m-““, such as kinase activity (Kloetzer et UC, 1983; 1984), would also be expected to increase as the level of this protein increases. The results presented here provide further support for our model by demonstrating that 28” is the optimal temperature for synthesis of P8F-““, and that lowering the temperature from 33 to 28” induces a corresponding increase in P85gW-ma8, in the mRNA for P85W+-, the kinase activity associated with this protein, and the morphological transformation of the culture, while corresponding decreases in the levels of P58g”8 and its mRNA are observed. METHODS

CelLs and virus. tsll0 Moloney murine sarcoma virus (MO-M&V) was derived

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from Mo-MuSV-349, a subclone of MoMuSV-124 (Ball et d, 1973), as described by Blair et al. (1979). Nonproducer normal rat kidney (NRK) cells infected with tsll0 MO-MuSV, termed 6m2 (Blair et al, 1979), were maintained in McCoy’s 5a medium containing 15% (v/v) fetal calf serum. Protein labeling and cktection. 6m2 cells were pulse-labeled with L+%I]leucine (high specific activity, New England Nuclear Corp.) at 500 &i/ml in Earle’s balanced salt solution for 20 min at the temperature indicated in T-25 flasks. The cells were seeded at 33”, then maintained at various temperatures for at least 24 hr prior to pulse labeling. After cell lysis, the cytoplasmic extracts were immunoprecipitated with either anti-p10 or anti-p15 goat sera absorbed with uninfected mouse cell extracts prepared from purified Rauscher murine leukemia virus as described by Naso et al. (1975). For analysis of steadystate levels of P85 and P58, 6m2 cells were incubated for 3 hr in leucine-deficient MEM media containing 500 &i/ml pH]leutine and 15% dialyzed fetal calf serum (DFCS). In this case cell lysis and immunoprecipitation were done as described under the protein kinase section using anti-p30 serum. The sera were supplied by the Logistics Program of the National Cancer Institute. Indirect immunoprecipitation was performed using formalininactivated Staph&coccus aureus (Cowan strain) as described by Kessler (1975). The immunoprecipitates were washed and fractionated by SDS-polyacrylamide gel electrophoresis on 8% acrylamide gels (Arcement et aL, 1976) and the dried gels developed by fluorography as described (Jamjoom et al, 1977). Analysis of intracellular viral RNA. Total cellular RNA was extracted by the hot phenol procedure as previously described (Hamelin et al, 1973). (Poly)A-containing RNA was twice selected on (oligo)dT cellulose (Aviv and Leder, 1972), denatured in the presence of glyoxal (McMaster and Carmichael, 1977), and subjected to electrophoresis on 1% agarose gels at 50 V for 10 hr. The procedure of (Thomas, 1980) was used for transfer of RNA to nitrocellulose sheets and viral-specific RNA was

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revealed by hybridization with nicktranslated q-labeled DNA probes (Rigby et aL, 1977). Conditions of hybridization and washing have been described (Hamelin et uL, 1983). The mos-containing DNA probe utilized was a B&II-HindIII, 1.3-kb fragment of MO-MuSV 124 DNA inserted into pKC7 plasmid (Junghans et c& 1982). This fragment is not completely mos specific since it contains 174 bp of sequence information upstream from the v-mos gene. Protein kinase assag. This assay was performed by a modification of the kinase assay as previously described (Kloetzer et a!, 1983). Cells grown to 80% confluency in 25-cm2 flasks were thoroughly drained and residual media removed with a Pasteur pipette. The flask was placed on ice and the cells were suspended in 0.5 ml of an NP-40 lysis buffer (1% NP-40,150 mM NaCl, 1 mMEDTA, 100 KIU of aprotinin/ ml, 10 mM sodium phosphate, pH 7.2) with the aid of a cell scraper. After further disruption with a Wheaton type B Dounce glass homogenizer, the lysate was clarified by high-speed centrifugation for 30 min at 30,000 rpm (Beckman 50Ti rotor). The resulting supernatant fluid was absorbed with 0.2 ml of Pansorbin to reduce the nonspecific binding of cellular kinases (Kloetzer and Arlinghaus, 1982). Immune complexes were formed by incubating 0.5 ml of cell extract in a 5-ml culture tube with 0.01 ml of antiserum 2 hr on ice. An equal volume of Pansorbin was then added. After 20 min on ice, the immunoprecipitates were washed several times with PBS/NP-40 (0.1% NP-40, 150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2) and finally suspended in 0.05 ml PBS/NP-40. An equal volume of PBS/NP40 containing 15 mA4 MnC12, 10 PM ATP, and 0.01 mCi [T-~~P]ATP (4000-5000 Ci/ mmol, ICN) was added to initiate the reaction. A standard assay was incubated for 10 min at 22” with occasional swirling. The reaction was terminated by addition of 3 ml RIPA buffer containing 2 mM ATP buffer (1% NP-40, 0.1% SDS, 1% DOC, 0.15 M sodium phosphate buffer, pH 7.2, 125 KIU/ml Trasylol, 2 mM EDTA). The immune complexes were further washed twice with RIPA buffer, heated 3

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min in boiling water in an SDS+mercaptoethanol-containing sample buffer (40 /.d), and then spun 10 min at 5000 rpm (Sorvall H4000 rotor). The supernatant fluid was removed and applied to an 8% resolving gel for SDS-gel electrophoresis. RESULTS

Eflect of temperature variation on the expression of P8F”“. 6m2 cultures were grown at 33” until they were approximately 70% confluent; cultures were shifted for 24 hr at temperatures from 20 to 39”. In a typical experiment approximately equal numbers of cells were pulselabeled with L-pH]leucine for 20 min and the extracts were divided into two equal aliquots for immunoprecipitation with either anti-p10 or anti-p15 sera. SDS-PAGE analysis showed that P85gw”” and P58B”B synthesis varied with temperature (Fig. 1). At 39 and at 37” no P85g”B-“” was detected (lanes 10 and 12), in agreement with our previously published work (Horn et al, 1981), but in cells grown at 33” (lane 8), P858”9-“” was clearly detectable as expected. Interestingly, at 28” (lane 6) the amount of P85@‘-“” synthesis was dramatically increased while P588”8 synthesis was sharply decreased. The ratio of P85 to P58 at temperatures of 24 and 20” was variable possibly due to the very slow growth rate (and hence their metabolic rate) of cells at these temperatures. To determine the optimal permissive temperature for the synthesis of Ps5Bag-, we tested cells maintained for 24 hr at 33, 30, 28, 26, and 24’ (Fig. 2A). Figures 2B-E show the gel tracings of the results shown in Figure 2A. The results clearly show a shift in the ratio of P8E9”8-“” to P588”Q as the temperature is lowered from 33 to 28’. At 30” (Fig. 2C), a nearly equal rate of synthesis of P858”8-“” and P58w was observed. At 28” (Fig. 2D), the ratio of P858”g-- to P58m is greatest, and is approximately the inverse of that observed at 33” (Fig. 2B). Such a result is consistent with our proposed splicing mechanism for generating the P85 mRNA (3.5 kb) from the P58 mRNA (4.0 kb). Effect of tempe-rature on the RNA of

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FIG. 1. Variation of the synthesis of P8p.“” and P58W with temperature. 6m2 cells were maintained at various temperatures for 24 hr and pulse-labeled with Lj3H]leucine for 20 min. Cytoplasmic extracts were immunoprecipitated with either anti-p10 (lanes 1,3, 5, 7, 9, and 11) or anti-p15 (lanes 2, 4, 6, 8, 10, and 12). The washed immunoprecipitates were analyzed by SDSpolyacrylamide gel electrophoresis (8%) geI, dried, and processed for fluorography. Lanes 1, 2cells at 20°; lanes 3, 4-24”; lanes 5, 6-28”; 7, 8-33”; lanes 9, 10-37”; lanes 11, 12-39”.

MeMuSV 6m2 cells. In order to determine whether or not the ratio of tsll0 viral RNAs was affected by the

temperature at which 6m2 cells are maintained, we extracted poly(A)-containing RNA from 6m2 cells grown at 39, 37, 33,

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FIG. 2. Quantitation of the levels of PW-“” and P58°“g synthesized in 6m2 maintained at various temperatures. 6m2 cells were maintained for 24 hr at various temperatures, pulse-labeled with L-pH]leucine as described in Fig. 1. .Using fluorography, a preflashed X-ray film was exposed to the dried gel to produce a fluorogram that was scanned to determine the amounts of PW-“” and P58p”o. The upper left panel shows the portion of the fluorogram used to prepare the tracings shown in panels B-F. In the upper left panel, 6m2 ceils were pulse-labeled for 20 min and processed for immunoprecipitation with anti-p10 (the A lanes) or anti-p15 (the B lanes). Panel B shows the tracings from cells grown at 33’; panel C-30°; panel D-28”; panel E-26O; panel F-24”.

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and 28” and analyzed them by Northern blotting using a labeled v-mos DNA probe to detect tsll0 viral RNAs. The results are shown in Fig. 3. At 39” (lane 4) and 37’ (lane 3) only the 4.0-kb viral RNA was detected. At 33” (lane 2) small amounts of the 3.5-kb gw-wws mRNA was detected. However, at 28’ (lane 1) the ratio of the 3.5- to 4.0-kb RNA species was completely reversed compared to 33’, i.e., a large increase in the 3.5-kb RNA was observed at 28”, and this increase corresponded with a decrease in the 4.0kb RNA. These results parallel those observed for the P5Sm and P87, and provide further support that the 3.5-kb viral RNA is derived from the 4.0-kb species, although other interpretations can be made from these results such as rapid turnover of the 3.5-kb RNA at temperatures of 33” and above. E#ect of temperature on the wwqhology of 6m2 cells. We have previously reported that 6m2 cells undergo a reversible change in shape and morphology at temperatures between 33 and 39” (Horn et aL, 1981; Brown et aL, 1981; Stanker et d, 1983b). Since the amount of P858”8-“” is sign& cantly increased when the temperature at

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FIG. 3. Northern blot analyses of 6m2 cells maintained at various temperatures. 6m2 cells were maintained at 23’ (lane l), 33” (lane 2), 37” (lane 3), and 39” (lane 4) for at least 24 hr and (poly)Acontaining RNA was extracted. (Po1y)A RNA was separated by electrophoresis, blotted onto nitrocellulose, and hybridized with a labeled v-mea probe. Different levels of RNA were applied to each lane: lane 1 (-2 pg), lane 2 (-6 m), lane 3 (-4 pg), and lane 4 (-6 pg).

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which cells are grown is shifted from 33 to 28”, a similar effect in the transformed phenotype would be expected under these conditions. The results (Fig. 4) show a significant increase in cell transformation as judged by the frequency of rounded cells in cultures maintained at 28” (Fig. 4A) when compared to culture maintained at 33’ (Fig. 4B). Cells maintained at 39” (Fig. 4D) were flat as expected. Cell maintained at 28” are viable but grow more slowly than at 33”. Eflect of temperature on the gag-mosassociated pro&h kinase. We have shown that P858”g” has an associated protein kinase activity which phosphorylates itself and P588”8 on serine and threonine residues (Kloetzer et al, 1983). Furthermore no kinase activity is detectable in immunoprecipitates obtained from 6m2 cells maintained at 39”. Such immunoprecipitates from 6m2 cells maintained at 39” contain relatively large amounts of P588”g, even though this protein is not phosphorylated during the test tube incubation. Thus, the kinase activity appears to depend upon the presence of P85Q”Q-“” in the immunoprecipitates. To determine if kinase activity corresponded to levels of P85g”B-““, the steady-state levels of this protein were compared to immune complex kinase activity in 6m2 cultures maintained at various temperatures. Figure 5 shows the results of the immune complex kinase assay from 6m2 cells grown at 28, 33,37, and 39” (lanes 2,4, 6, and 8 of Fig. 5). The kinase activity was found to increase when derived from 6m2 cells maintained at 28” (lane 2) compared to 33” (lane 4) as determined by P858”8-“” phosphorylation. Quantitation of the bands by scanning the gel showed a four- to fivefold increase in P85 phosphorylation. This result agreed with the measured increase in the steady-state level of PSp”* seen at these temperatures (Fig. 5, lanes 1, 3, 5, and 7). Furthermore, in vitro phosphorylation of P5w also increased as the temperature was decreased from 37 to 28O, even though the steady-state level of this protein decreases. These results show that P858”9”“, not P5gm, is associated with the kinase activity. One difference observed between the results of steady-

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85,000-Da gag-mos hybrid protein, termed PSF--, allows for studies of functions which correlate with synthesis of this protein. In this report we have shown that both the Ps58”6-“” transforming protein and the mRNA encoding this protein P85*-vary with the temperature at which the cells are maintained. In addition, the concentration of P85B”Q-“” directly correlates with the kinase activity associated with P85. Thus, our results show that a fourto fivefold increase in P85 results in a similar increase in the activity of the kinase associated with P85. We have proposed that the tsll0 virus contains a temperature-sensitive defect FIG. 5. In vitro kinase activity associated with that affects transcription of the gag-mos P8W”” from 6m2 cells maintained at various temperatures. Identically seeded 25-cm” flasks of 6m2 mRNA (Horn et d, 1981). We have further proposed a model whereby the 4.0-kb gecells were grown to 80% conffuency at 33“ and then incubated at 28, 33, 37, and 39” for 16 hr. Lanes 1, nomic RNA (from which the truncated is spliced 3, 5, and 7 are anti-p30 immunoprecipitates from gag protein, P588”8 is translated) to yield the 3.5-kb mRNA which is trans6m2 cells incubated at 23,33,37, and 39’, respectively, that were labeled for 3 hr with 500 &i/ml Llated to yield the gag-mos protein (Jun[*H]leucine in leucine-deficient MEM containing 15% ghans et aL, 1982). Recent results obtained dialyzed fetal calf serum. Lanes 2, 4, 6, and 8 are using the Sl method of analyses have anti-p30 immunoprecipitates from cells incubated at supported this interpretation (Nash et al, 28, 33, 37, and 39’, respectively, that were subjected 1984). Northern blot analyses shown here to the immune complex kinase assay using -r-=Pprovide direct proof that the 4.0-kb viral labeled ATP. genomic RNA is made at restrictive (39”) and permissive (33”) temperatures whereas the 3.5-kb RNA is detectable only state labeling (Fig. 5) and pulse-labeling The highest (Fig. 2) is the presence of P858”8-- (and at permissive temperatures. accumulation of the 3.5-kb RNA is at 28”, kinase activity) at 37”. This difference is and lowering the temperature from 33 to likely the result of a small amount of 28” results in a dramatic shift in the ratio P858”8-“” being synthesized at 37”, which of the amount of 4.0- to 3.5-kb RNA in can only be detected at prolonged labeling favor of the 3.5-kb RNA. The level of the periods. Thus, the level of Pw-“” di3.5and 4.0-kb RNAs are mutually affected rectly correlates with the level of its mRNA, its kinase activity, and the degree in the shift from 33 to 28”; the 4.0-kb RNA decreases whereas the 3.5-kb RNA of morphological transformation of tsllOincreases. Thus, these results are consisinfected cultures. tent with the interpretation that the 3.5kb RNA is made from 4.0-kb RNA by a DISCUSSION splicing event. However, other interpreStudies of transformation induced by tations are possible. For example, the 3.5murine sarcoma viruses have been greatly kb RNA may be heat sensitive and be complicated because of the trace amounts unstable at temperatures of 33” and above. of the enu-mos protein produced by pro- In addition, more than one provirus may totype Moloney MuSV strains. Such stud- exist, one for each of the viral RNAs. Differential transcription with temperaies are facilitated in the tsll0 Moloney MuSV system because of the relatively ture could then yield these results. Howabundant amount of gas-mos protein pro- ever, recent studies by our laboratory in duced in tsllO-infected cells. Further, the collaboration with E. C. Murphy, Jr. at temperature-sensitive production of an M. D. Anderson have recently provided 1

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evidence for only one tsll0 provirus in 6m2 cells (R. Hamelin, E. C. Murphy, Jr., and R. Arlinghaus, ViroZoga, in press). Thus, both tsll0 RNAs appear to arise from a single provirus. The shift in the ratio of the 4-kb RNA to 3.5kb RNA is also supported by the level of synthesis and steady-state concentration of P858”8-“” and P588”8 in 6m2 cells. We have shown that P858”8-“” is made from the 3.5-kb RNA and P588”6 is made from the 4.0-kb RNA (Junghans et al, 1982). As expected then, at 33”, the amount of Ps5B”g-““” is much reduced when compared to that of P588”8 whereas at 28” the level of P85-“” increased dramatically, and P588”8 is reduced. The ability to alter the levels of transforming protein by temperature manipulation allows for examination of properties associated with synthesis of this protein. For example, we have shown that P858”9”* has a kinase activity that is either an intrinsic property of the protein itself or one that is closely associated with the 1120sportion of P85-“” (Kloetzer et aL, 1983,1984). The data presented here demonstrate that the amount of P858”Q-“” kinase activity closely correlates with the concentration of P858”8-“” in 6m2 cells. Furthermore the level of P85@‘@“” correlates with the degree of morphologic transformation of 6m2 cells. Cells are markedly more rounded at 28” than at 33”. Thus, by manipulation of the temperature at which tsllO-infected cells are grown, the degree of transformation can be controlled and studied. This system offers future potential for understanding mechanisms of splicing, as well as for examining properties and effects of the gag-mos protein such as substrates for the associated kinase, and binding lipid (Gallick and Arlinghaus, 1984), all of which may be important in developing an understanding of the mechanism of transformation by v-mos. Such investigations are currently in progress. ACKNOWLEDGMENTS Part of this research was performed while R.B.A. was a staff member of the University of Texas M. D. Anderson Hospital and Tumor Institute in Hous-

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ton, Texas 77030. During that time this research was supported in part by a grant from the Robert A. Welch Foundation (G-429), Public Health Service Grant CA-25465, and Core Grant CA-16672. G.E.G. was supported by Public Health Service Training Grant CA-09299; R.H. was partly supported by the Ligue Nationale Francaise Contre le Cancer and the Fondation pour la Recherche Medicale; S.M. received fellowship support from the Rosalie B. Hite Foundation. REFERENCES ARCEMENT, L. J., KARSHIN, W. L., NASO, R. B., JAMJOOM, G. A., and ARLINGHAUS, R. B. (1976). Biosynthesis of Rauscher leukemia viral proteins: Presence of p30 and envelope p15 sequences in precursor polyproteins. Vi69, 763-774. AVIV, H., and LEDER, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligo thymidylic acid cellulose. Proc. Nati Acad Sti USA 69,1408-1412. BALL, J. K., MCCARTER, J. A., and SUNDERLAND, S. M. (1973). Evidence for helper independent murine sarcoma virus I. Segregation of replicationdefective and transformation-defective viruses. Virologg 56,120-H%. BLAIR, D. G., HULL, M. A., and FINCH, E. A. (1979). The isolation and preliminary characterization of temperature-sensitive transformation mutants of Moloney sarcoma virus. k?rology 95, 303-316. BROWN, R., HORN, J. P., WIEBLE, L., ARLINGHAUS, R. B., and BRINKLEY, B. R. (1981). Analyses of the sequence of events in the transformation process in cells infected with a ts transformation mutant of Moloney murine sarcoma virus. Proc NatL Acad

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GALLICK, G. E., and ARLINGHAUS, R. B. (1984). Incorporation of lipids into variants of Moloney sarcoma virus which produce gag-mos fusion proteins. virdogy 133. 228-232. HAMELIN, R., DEVAUX, J., HONORE, N., AUGER-BUENDIA, M. A., and TAVITIAN, A. (1983). Characterization of viral RNA in cells transformed by various isolates of Moloney murine sarcoma virus. J. Gen Vird 64, 2057-2062. HAMELIN, R., LARSEN, C. J., and TAVITIAN, A. (1973). Effects of Actinomycin D Toyocamycin and cycloheximide on the synthesis of low molecular weight nuclear RNA in HeLa cells. Eur. J. Biochem 35, 350-356. HORN, J. P., WOOD, T. G., MURPHY, E. C., JR., BLAIR, D. G., and ARLINGHAUS, R. B. (1981). A selective temperature-sensitive defect in viral RNA. Expression in cells infected with a ts transformation mutant of murine sarcoma virus. CeU 25. 37-46. JAMJOOM, G. A., NASO, R. B., and ARLINGHAUS, R. B. (1977). Further characterization of intracel-

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lular precursor polyproteins of Rauscher leukemia virus. Vir& 78.1134. JUNGHANS, R. P., MURPHY, E. C., JR., and ARL.INGHAUS, R. B. (1982). Electron microscopic analysis of tall0 Moloney murine sarcoma virus, a variant of wild-type virus with two RNAs containing large deletions. J. MOL Bid 161, 229-255. KESSLER, S. W. (1975). Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: Parameters of the interaction of antibody-antigen complexes with protein A. J. Immunol 115, 1617-1624. KLOETZER, W., and ARLINGHAUS, R. (1982). Binding of retrovirus-associated protein kinase and proteins to Staphylococcus aureus. J. Vird 60,365-370. KLOETZER, W. S., MAXWELL, S. A., and ARLINGHAUS, R. B. (1983). P&Y@‘++ encoded by tsll0 Moloney murine sarcoma virus has an associated protein kinase activity. Proc. NatL Acad Sci USA 80,412416. KLOETZER, W. S., MAXWELL, S. A., and ARLINGHAUS, R. B. (1984). Further characterization of the P&Z+“‘@ ““-associated protein kinase activity Virdogy 138, 143-155. MCMASTER, G. K., and CARMICHAEL, G. C. (1977). Analysis of single and double stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74,4835-4838.

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ET AL. R. B., and MURPHY, E. C., JR. (1984). Sl nuclease mapping of viral RNAs from a temperature-sensitive transformation mutant of murine sarcoma virus. J. Viral 50,478-488. NASO, R. B., ARCEMENT, L. J., and ARLINGHAUS, R. B. (1975). Biosynthesis of Rauscher Leukemia viral proteins. CeU 4, 31-36. PAPKOFF, J., VERMA, I. M., and HUNTER, T. (1982). Detection of a transforming gene product of Moloney murine sarcoma virus in transformed cells. Cell 29,417-426. RIGBY, P. W. J., DIECKMANN, M., RHODES, C., and BERG, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Bid 113,237-251. STANKER, L. H., HORN, J. P., GALLICK, G. E., KLOETZER, W. S., MURPHY, E. C., JR., BLAIR, D. G., and ARLINGHAUS, R. B. (1983a). gag-wws polyproteins encoded by variants of the Moloney strain of mouse sarcoma virus. Virology 126, 336347. STANKER, L. H., GALLICK, G. E., HORN, J. P., and ARLINGHAUS, R. B. (1983b). P85-“” encoded by tsll0 Moloney murine sarcoma virus: Rapid turnover at the restrictive temperature. J. Gen FiroL 64,2203-2211. THOMAS, P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci USA 77,52015205.