Vol. 16, No. 3 Printed in U.S.A.
INFwCTION AND ImmuNrry, June 1977, p. 742-747 Copyright © 1977 American Society for Microbiology
Synthesis and Cleavage Processing of Oncornavirus Proteins During Interferon Inhibition of Virus Particle Release STUART Z. SHAPIRO, MElTE STRAND, AND ALFONS BILLIAU* Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, and Department ofHuman Biology, Rega Institute for Medical Research, University ofLeuven, Leuven, Belgium* Received for publication 24 November 1976
The effect of interferon on the rate of synthesis and the cleavage processing of viral proteins in mouse cells, chronically infected with Rauscher murine leukemia virus, has been studied by immunoprecipitation of newly synthesized viral proteins from virus-infected cells pulse-labeled with [35S]methionine. Immunoprecipitated, labeled polypeptides were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and then examined by autoradiography. Cleavage processing was studied in the same manner with cells that had been pulse-labeled and then incubated with non-radioactive media for a sufficient time to allow normal cleavage processing to occur. At a concentration that strongly inhibited the release of virus particles, interferon had no effect on the synthesis of proteins carrying antigenic determinants of the major core protein p30 or of the envelope glycoprotein gp69/71. Nor did it affect the post-translational cleavage processing of the precursors to these proteins. Similarly, interferon did not affect labeling or chasing of precursor protein carrying the p15 deterninants; labeling of p15 itself could not be studied because it does not contain methionine.
Interferon has been shown to inhibit the production of type C oncornavirus particles in chronically infected cells (1-5, 7, 19). The mechanism of this inhibition is not known. It has been postulated that interferon inhibits cytolytic virus replication by interfering with translation of viral proteins (16). However, evidence exists that this may not apply to oncornaviruses. Oncornavirus-infected cells, which undergo multiple divisions in the presence of interferon, contain unaltered or increased, rather than decreased, levels of viral proteins (7, 8), and interferon-treated cells show a larger number of membrane-associated virus particles than control cells (2-4). These findings have led to the suggestion that interferon might inhibit type C virus release instead by interfering with some late step in particle assembly or release (2, 7, 8). One limitation to this model is that as yet, only the cellular concentration of viral proteins has been measured, and no distinction has been made between the rate of synthesis of viral proteins and the accumulation of viral proteins in the cell or in cell-associated virus particles. Thus, it is possible that the rate of synthesis of the major viral proteins is decreased by interferon but the proteins are retained by the cells, thereby effectively masking the decreased rate of synthesis. The present studies were designed to directly analyze the
synthesis of specific virus proteins in interferon-treated cells and, in addition, to ascertain whether interferon treatment had any effect on the post-translational processing of the major viral proteins that occurs during virus particle assembly.
742
MATERIALS AND METHODS Cell cultures and virus. NIH/3T3 cells were a gift from A. Hackett (Cell Culture Laboratory, Naval Biomedical Research Laboratory, Oakland, Calif.). The Rauscher murine leukemia virus-infected NIH/ 3T3 cell line used in these studies was established by infection of NIH/3T3 cells with Rauscher virus propagated in a normal rat kidney cell line obtained from E. Scolnick (National Institutes of Health, Bethesda, Md.). All cells were propagated at 37°C in Dulbecco-modified Eagle medium supplemented with 10% fetal bovine serum (Flow Laboratories, Rockville, Md.), penicillin (50 ,ug/ml), streptomycin (50 ,ug/ml), neomycin (100 utg/ml), and an organic buffer system {HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 7.5 mM; MOPS [3-Nmorpholinepropanesulfonic acid], 5 mM; TES [N-
tris(hydroxymethyl)methyl-2-aminomethane-sulfonic acid], 5 mM} described by Eagle (6). Unlabeled Rauscher virus propagated in a mouse bone marrow cell line, JLSV9 (22), was provided by the John L. Smith Memorial for Cancer Research under the auspices of the Special Virus Cancer Program, National Cancer Institute. Antisera. Monospecific antisera against three purified structural proteins of Rauscher virus, gp69/
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INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION
71, p3O, and p15, was prepared as described elsewhere (16). Examination of each antiserum by the competition radioimmunoassay for reactivity toward the other viral proteins indicated no detectable antigenic cross-reactivity. Cellular proteins coprecipitated with viral proteins in the immunoprecipitation procedure are distinguished from viral specific proteins by control immunoprecipitation reactions using uninfected cells (14). Interferon. Interferon was prepared from mouse L-929 cells by infection with Newcastle disease virus as described elsewhere (5). The preparation used in the present study contained 1045 NIH reference units/ml. It contained 10% fetal bovine serum from the culture medium, so that the specific activity can be estimated to be around 103 5 reference units/mg of protein. In control experiments on JLSV5 cells, doses of 100 reference units or less had no measurable effect on cell growth. At 300 and 1,000 reference units/ml cell growth was slightly inhibited. Interferon sensitivity assay. The inherent sensitivity of cell lines to the antiviral action of interferon was tested by a vesicular stomatitis virus (VSV) yield reduction assay as described earlier (1). Briefly, cell monolayers were exposed to serial dilutions of interferon for 24 h. The cultures were then drained and infected with VSV at a multiplicity of infection of 1. After incubation for 1 h, excess virus was removed by washing, fresh medium was added, and incubation was continued for 16 h. Virus was harvested and titrated by plaque assay on L-929 cells. The sensitivity of a cell line to interferon is indicated by the concentration of interferon necessary to inhibit the VSV yield by a factor of 3 (0.5
log10).
Reverse transcriptase assay. Culture supernatants were processed for reverse transcriptase determination as follows. After clarification by low-speed centrifugation, the virus in each sample was pelleted by centrifugation at 100,000 x g for 60 min, suspended in NT buffer [100 mM NaCl, 10 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.4], sedimented again as above, and finally resuspended in 0.5 ml of NT buffer. Samples of 75 ,ul were mixed with an equal volume of 100 mM Tris-hydrochloride (pH 8.3) containing 0.4% Triton X-100; 25-Il samples of this disrupted virus were assayed in reaction mixtures containing 0. 1% Triton X-100, 50 mM Tris-hydrochloride (pH 8.3), 100 mM NaCl, 0.5 mM manganous acetate, 5 mM dithiothreitol, 1 ,uM [3H]deoxythymidine 5'-triphosphate (approximately 30,000 cpm/pmol), 1.9 AM oligodeoxythymidylic acid [oligo(dT)12-18], and 7.5 ,uM polyriboadenylic acid [poly(rA)] or 7.5 itM polydeoxyadenylic acid [poly(dA)] in a total volume of 100 ,ul. Reaction mixtures were incubated for 60 min at 35°C, 100 ,ug of yeast ribonucleic acid (RNA) was added as carrier, and the reaction was terminated by the addition of 10 volumes of TP solution (5% [wt/ wt] trichloroacetic acid and 20 mM sodium pyrophosphate). Acid-insoluble material was collected on membrane filters, washed exhaustively with TP solution and then twice with ethanol, and heatdried, and the radioactivity was counted in a toluene-based scintillant. All values were the average of
743
duplicate reactions. Each sample was also tested for the presence of inhibitory activity toward reverse transcriptase by adding 25 pl of the disrupted virus preparations to standard reaction mixtures containing purified Rauscher virus and the poly(rA) template. No inhibitory activity was found in any of these samples. The activity of the samples in reaction mixtures containing poly(rA) as template was at least 50-fold the amount of [3H]deoxythymidine 5'-triphosphate incorporated by samples in reaction mixtures containing poly(dA). Assay of virus production by [3H]uridine incorporation. Cell cultures previously treated for 24 h with interferon and parallel control cultures were incubated for 24 h with growth medium containing the appropriate concentration of interferon and [3Hluridine (39.3 Ci/mmol, New England Nuclear Corp., Boston, Mass.) at 250 ACi/75-cm2 cell culture flask (T75 flask, Falcon Plastics, Oxnard, Calif.). The supernatant fluid was harvested, clarified by low-speed centrifugation, concentrated by treatment with dry Ficoll (Pharmacia Fine Chemicals, Piscataway, N.J.), and dialyzed against 25 volumes of TEN buffer (20 mM Tris-hydrochloride, pH 7.4, 1 mM ethylenediaminetetraacetic acid, and 100 mM NaCl). The preparations were layered on top of 11ml sucrose gradients (20 to 50%, wt/wt, in TEN buffer) and centrifuged for 18 h at 30,000 rpm (SW41 rotor, Beckman Instruments, Fullerton, Calif.). The gradients were fractionated into 0.5-ml samples, and acid-precipitable radioactivity was measured. Type C oncornavirus bands at 1.16 to 1.18 g/cm3 in sucrose density gradients. Analysis of viral protein synthesis by immunoprecipitation. Cultures previously treated for 24 h with interferon (1,000 U/ml) and analogous control cultures were incubated for 2 h at 37°C with 10 ml of serum-free minimal essential medium lacking methionine. The medium was then replaced with 3 ml of the same methionine-free medium plus 250 ,uCi of [35S]-methionine per ml (about 300 Ci/mmol, Amersham-Searle, Arlington Heights, Ill.), and incubation was continued for 20 min. Cells were either lysed immediately after labeling, or the labeling medium was replaced with non-radioactive growth medium, and incubation was continued for 3 h before lysis. The concentration of interferon with which each culture had been pretreated was maintained during prelabeling, labeling, and chase incubations. Cell protein was solubilized by incubation of the cells in each T75 flask at 37°C for 15 min with 2 ml of extraction buffer, 5 mM Tris-hydrochloride (pH 9.2) containing 1% Triton X-100, 400 mM KCl, 1 mM ethylenediaminetetraacetic acid, 1 mM L-1-tosylamide phenylethylchloromethyl ketone (Sigma Chemical Co., St. Louis, Mo.), and 1 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co.). The suspension was centrifuged at 27,000 x g for 10 min. The supernatant was retained. The pellet was suspended in 1 ml of extraction buffer lacking KCl and incubated for 15 min at 37°C. This suspension was centrifuged as above, and the supernatant was collected. The pooled supernatants were twice dialyzed for 3 h against 25 volumes of TEN buffer followed by centrifugation for 1 h at 100,000 x g.
744
SHAPIRO, STRAND, AND BILLIAU
Labeled polypeptides carrying virus protein antigenic determinants were immunoprecipitated by addition of 25 gl of anti-gp69/71 serum, 30 ,ul of antip30 serum, or 50 /.l of anti-p15 serum plus 5 pg of unlabeled Rauscher virus as carrier to 1 ml of labeled cell extract, followed by incubation overnight at 40C. The precipitate was collected by centrifugation for 30 min at 4,500 x g, washed twice with TEN buffer and once with acetone, and then dissolved in electrophoresis sample buffer. Electrophoresis was performed in a high-resolution 5 to 20% gradient polyacrylamide slab gel in the presence of 0.1% sodium dodecyl sulfate as described by Maizel (11). Slab gels were dried, and autoradiography was performed with Kodak No Screen medical X-ray film (NS54T). Radiolabeled protein was measured by scanning the autoradiographs in a Joyce-Loebl microdensitometer.
INFECT. IMMUN.
tures were then challenged with the cytolytic virus VSV as outlined in Materials and Methods. Analogous cultures were washed, and incubation with the appropriate concentration of interferon was continued for 24 h, after which oncornavirus release was determined by measurement of reverse transcriptase released into the medium. Inhibition of replication of both VSV and type C virus by interferon was observed to be dose dependent (Fig. 1). Approximately 3 reference units of interferon per ml were necessary to inhibit the yield of VSV to 30% of the control value, whereas approximately 16-fold more, i.e., 50 reference U/ml, was necessary to inhibit release of particleassociated reverse transcriptase to the same extent. Similar ratios for sensitivity of VSV and type C virus were found in JLSV5 and RESULTS cells (1-5). Sensitivity of Rauscher virus-infected NIH/ MO-P Experiments for determination of oncornavi3T3 cells to interferon. A cell line (NIHI3T3) rus protein synthesis were performed with a that is relatively noninducible for endogenous great excess of interferon (i.e., 1,000 reference oncornavirus production (10) was chosen for To confirm that this dose of interthese experiments to avoid the possibility of units/ml). effectively blocked release of virus partiendogenous oncornavirus protein synthesis feron cles into the supernatant fluid, parallel culmasking an interferon effect on the synthesis of tures were incubated with 0 or 1,000 reference Rauscher virus proteins. The interferon-inof mouse interferon for 24 h, followed by duced inhibition of both the replication of a units incubation for an additional 24 h with medium cytolytic virus and of the release of Rauscher containing the same concentration of interferon virus from chronically infected cells was exam- plus [3H]uridine. The medium was then asined. sayed for reverse transcriptase activity and for Confluent monolayers of Rauscher virus-in- 3H-labeled particles. Reverse transcripfected NIH/3T3 cells were incubated for 24 h tase activityvirus from interferonwith serial dilutions of mouse interferon. Cul- treated cells in media samples (1,315 cpm) was 5% of the activity in equivalent samples from untreated Rauscher virus-infected NIH/3T3 cells (26,313 cpm). Uninfected cells showed insignificant incorpora100 tion (60 cpm). Sedimentation in a sucrose density gradient of supernatant fluid from an untreated Rauscher virus-infected culture yielded 0 3030 a peak of acid-precipitable radioactivity at 0 1.165 g/cm3 (6,544 cpm), whereas sedimentation 0 of fluid from a parallel interferon-treated culture failed to produce a distinct peak; acid0 \ precipitable radioactivity banding at 1.165 g/ w cm3 was 3,364 cpm with treated cultures and 3 5 2,200 cpm when fluids from uninfected NIH/3T3 cells were tested. It was thus concluded that Rauscher virus> I infected NIH/3T3 cells respond to interferon in much the same way as previously examined 0 1 100 1000 10 cell lines, such as JISV5 and MO-P (5), and INTERFERON (units/ml) that the dosage used in experiments measuring FIG. 1. Production ofRauscher virus and VSV in viral protein synthesis effectively reduced the Rauscher virus-infected NIH/3T3 cells exposed to in- release of type C virus particles into the superterferon for 48 h. Symbols: (-) 24- to 48-h yield of natant fluid. Effect of interferon on viral protein syntheVSV as determined by plaque assay on L-929 cells; (0) 24- to 48-h yield ofRauscher virus as determined sis and processing. Several of the structural by reverse transcritase assay. proteins of Rauscher virus have been shown to
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INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION
be synthesized first as higher-molecular-weight precursors, which are then cleaved during virus assembly and maturation to produce the mature virus proteins (for a review, see reference 13). The Rauscher virus envelope glycoprotein gp69/71 is the cleavage product of a glycosylated 90,000-dalton polypeptide, Pr9O. The p30 and p15 proteins of the virus are both cleavage products of the same 65,000-dalton precursor polypeptide, Pr65. The effect of interferon on the synthesis of these virus protein precursors was examined. Parallel uninfected NIH/3T3 and Rauscher virus-infected NIH/3T3 cell cultures grown to confluence were incubated for 24 h with either 0 or 1,000 reference units of mouse interferon per ml. The cultures were then washed, pulse-labeled with [35S]methionine, and processed for analysis of the synthesis of gp69/71 precursor Pr9O by immunoprecipitation with anti-gp69/71 serum (Fig. 2). After a 20-min pulse-label, more than 95% of the precipitated viral specific label was in the 90,000-dalton precursor polypeptide (Fig. 2A). Comparison ofthe amount of label in virus-specific protein peaks from treated and untreated cultures indicated that the rate of synthesis of this precursor protein was not affected by interferon treatment. A larger protein weighing 145,000 daltons, which is possibly a precursor to Pr9O, is also observable in Fig. 2A. During a 3-h chase, the 145,000-dalton polypeptide disappeared, and the concentration of Pr9O decreased as the virion envelope glycoprotein gp69/71 appeared in the cell extracts of both treated and untreated cultures (Fig. 2B). The presence of interferon had no apparent effect on the cleavage processing of the precursors to gp69/71. Analogous results demonstrating a lack of effect on protein synthesis and processing by interferon were obtained from immunoprecipitations using anti-p30 serun (Fig. 3). After a 20-min pulse-label, the predominant virus-specific protein precipitable with both sera is a 65,000-dalton precursor polypeptide (Pr65). The concentration of this precursor appeared to be the same in treated and untreated cells. A larger precursor of 76,000 daltons (Pr76) and a possible precursor weighing 145,000 daltons are also observable in Fig. 3A. Some p30 protein was already seen in a 20-min pulse-label in both interferon-treated and untreated cells. With chase, the precursor proteins were processed at similar rates, and increased amounts of virion-sized p30 protein appeared in both treated and untreated cultures (Fig. 3B). In similar experiments using an anti-p15 serum and [35S]methionine the labeling and chasing of high-molecular-weight precursor pro-
745
B
B
z
FIG. .
tig69/7
ermgp69/71
0 U)
4
30 145 90 70 MOLEULARWEIGHT x 10-3
FIG. 2. Anti-gp69/71 serum precipitates from extracts of cells pulse-labeled with [5SJmethionine. Immunoprecipitated viral specific protein synthesized in ) or absence (-----) of interferon was the presence ( resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by autoradiography. Densitometer tracings of individual lanes of the autoradiogram are depicted. (A) 35S-labeled polypeptides immunoprecipitated from extracts of Rauscher virus-infected NIHI3T3 cells pulse-labeled for 20 min. (B) 35S-labeled polypeptides immunoprecipitated from extracts of Rauscher virus-infected NIHI 3T3 cells pulse-labeled for 20 min and then chased for 3 h prior to extraction. (C) 3"S-labeled cellular polypeptides non-immunospecifically coprecipitated with unlabeled antigen immunoprecipitated from an extract of labeled, uninfected NIHI3T3 cells.
teins was also unaffected by interferon treatment. Since p15 itself does not contain methionine, the complete conversion of the precursors to p15 cannot be observed in experiments that use methionine as the labeling amino acid (15). However, lack of effect on precursor labeling and chasing makes it seem unlikely that interferon treatment affects synthesis or processing of p15.
746
SHAPIRO, STRAND, AND BILLIAU
INFECT. IMMUN.
cultures (Fig. 3B). Also, a slight increase in the amount ot labeled gp69/71 in treated cultures was noted (Fig. 2B).
l z 0 C,)
145 76 65 MOLECULAR WEIGHT
30 x
10'3
FIG. 3. Anti-p30 serum precipitates from extracts of cells pulse-labeled with [35S]methionine. Immunoprecipitates were prepared and processed as described in the legend to Fig. 2. Protein was immunoprecipitated from Rauscher virus-infected cells pulselabeled for 20 min (A) or pulse-labeled for 20 min followed by a 3-h chase prior to extraction (B). An immunoprecipitate from an extract of labeled, uninfected NIH/3T3 cells (C) is included as a control. Symbols: ( ) protein synthesized in the presence of interferon; (-----) protein synthesized in the absence of interferon.
Since the rate of synthesis of viral proteins appeared to be unaffected while virus particle release from the cell was inhibited, an accumulation of cell-associated virus proteins was expected. Indeed, such an effect on the viral p30 protein was previously detected by Friedman and Ramseur (8) using a radioimmunoprecipitation inhibition assay. In the present studies the amount of labeled p30 protein immunoprecipitated after a chase period of 3 h from interferon-treated cultures appeared to be about twice the amount precipitated from untreated
DISCUSSION These findings confirm earlier observations by Friedman and co-workers (7, 8) of continued synthesis of p30 in the presence of interferon. Moreover, the data suggest that interferon does not have any effect on the rate of p30 synthesis and that the synthesis of two other viral proteins, the envelope glycoprotein and the p15 protein, is likewise unaffected by the presence of interferon. Furthermore, the cleavage of viral protein precursors that occurs during virus particle production appeared to be unaffected by interferon, whereas at the same time particle release was effectively reduced. Several possible explanations exist for the interferon-induced inhibition of oncornavLrus production. One possibility is that the synthesis of an as yet unstudied viral protein is inhibited. Although this cannot be eliminated from consideration, the presently available data argue against it. The non-glycosylated viriorn proteins p30 and p15 are part of one large polypeptide precursor, Pr65, which also appears to contain the p12 protein (17) and may contain the only other non-glycosylated major structural protein of the virus, plO (13). Failure of interferon to affect synthesis of p30 and p15 therefore implies failure to inhibit synthesis of the precursor of all non-glycosylated major structural proteins of the virus. The synthesis of the other major structural protein, the viral envelope glycoprotein gp69/71, is also observed to be unaffected by interferon. The work of Friedman et al. (7) suggests that the synthesis of the only remaining known viral protein, reverse transcriptase, is likewise unaffected by interferon. Thus, there does not appear to be an interferon-induced inhibition of synthesis of any of the known oncornavirus structural proteins. Other possible explanations of the interferon effect on oncornaviruses include an interferoninduced block of some step in virion assembly or release. Such a mechanism could involve either virus protein or the virus RNA. The data presented here suggest that the cleavage processing of any of the three virion major structural proteins studied is the step at which such a block occurs. However, not all of the cleavages of virion proteins have been examined. Cleavages should occur in the production of the smallest-molecular-weight structural proteins, p12 and plO. Also, recent evidence suggests that the reverse transcriptase of mammalian oncornaviruses is the cleavage product of a
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INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION
higher-molecular-weight protein (9). These unstudied cleavage events could provide steps in virion production where interferon could act. Another, as yet unstudied question is whether the glycosylation ofthe viral envelope glycoprotein is affected by interferon treatment. When glycosylation is inhibited by 2-deoxy-i-glucose, a 70,000-dalton polypeptide is observed in place of the glycoprotein precursor Pr9O (15). An inhibition of glycosylation by interferon should likewise have been reflected by a difference in the molecular weight of the envelope protein, its glycoprotein precursor Pr9O, or both proteins, and this was not observed (Fig. 2). However, changes in sugar residues might not have been detected. Alternatively, the mechanism of interferon inhibition could be in blocking the integration of a synthesized and fully processed viral protein into budding viral particles. It should be noted that interference with cleavage, glycosylation, or integration of viral proteins would be entirely novel mechanisms of interferon action. Classically, interferon has been thought to inhibit viral production by preventing viral messenger RNA translation. It has been postulated that interferon induces the synthesis of a cell protein(s), which interferes with the ability of viral messenger RNA to combine with ribosomes (16). An interferoninduced interaction with oncornaviral RNA, preventing proper packaging into particles, might therefore be a model of interferon inhibition of oncornavirus production that more closely parallels the effect of interferon observed in other virus systems. ACKNOWLEDGMENTS This investigation was supported by Public Health Service Training Grant 5T5 GM 1674 and Grant GM 11301-11 from the National Institute of General Medical Sciences, by Public Health Service Contract 71-2251 within the virus Cancer Program of the National Cancer Institute, and by the Belgian A.S.L.K. Cancer Foundation. The technical assistance of R. Conings and Francine Cornette and the editorial help of Janine Putzeys are gratefully acknowledged. We also thank J. Thomas August for his encouragement, guidance, and assistance.
LITERATURE CITED 1. Billiau, A., V. G. Edy, E. De Clercq, H. Heremans, and P. De Somer. 1975. Influence of interferon on the synthesis of virus particles in oncornavirus carrier cell lines. III. Survey of effects on A-, B- and C-type oncornaviruses. Int. J. Cancer 15:947-953. 2. Billiau, A., V. G. Edy, H. Sobis, and P. De Somer. 1974. Influence of interferon on virus-particle synthesis in oncornavirus carrier lines. II. Evidence for a direct effect on particle release. Int. J. Cancer 14:335340. 3. Billiau, A., H. Heremans, and P. T. Allen. 1976. Trapping of oncornavirus particles at the surface of interferon-treated cells. Virology 3:537-542. 4. Billiau, A., H. Heremans, P. T. Allen, and P. De Somer. 1975. Influence of interferon on the synthesis of
5.
6.
7.
8.
9. 10. 11.
12. 13. 14.
15.
16.
17.
18.
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virus particles in oncornavirus carrier lines. IV. Relevance to the potential application of interferon in natural infectious diseases. J. Infect. Dis. 133(Suppl.):51-55. Billiau, A., H. Sobis, and P. De Somer. 1973. Influence of interferon on virus particle formation in different oncornavirus carrier cell lines. Int. J. Cancer 12:646653. Eagle, H. 1971. Buffer combinations for mammalian cell culture. Science 174:500-503. Friedman, R. M., E. H. Chang, J. M. Ramseur, and M. W. Myers. 1975. Interferon-directed inhibition of chronic murine leukemia virus production in cell cultures: lack of effect on intracellular viral markers. J. Virol. 16:569-574. Friedman, R. M., and J. M. Ramseur. 1974. Inhibition of murine leukemia virus production in chronically infected AKR cells: a novel effect of interferon. Proc. Natl. Acad. Sci. U.S.A. 71:3542-3544. Gerwin, B. I., S. G. Smith, and P. T. Peebles. 1975. Two active forms of RD-114 virus DNA polymerase in infected cells. Cell 6:45-52. Levy, J. A. 1973. Xenotropic viruses: murine leukemia viruses associated with NIH Swiss, NZB and other mouse strains. Science 182:1151-1153. Maizel, J. V., Jr., 1971. Polyacrylamide gel electrophoresis of viral proteins, p. 179-246. In K. Maramoroach and H. Koprowski (ed.), Methods in virology, vol. 5. Academic Press Inc., New York. Naso, R. B., L. J. Arcement, and R. B. Arlinghaus. 1975. Biosynthesis of Rauscher leukemia viral proteins. Cell 4:31-36. Shapiro, S. Z., and J. T. August. 1976. Proteolytic cleavage events in oncornavirus protein synthesis. Biochim. Biophys. Acta 458:375-396. Shapiro, S. Z., and J. T. August. 1976. The use of immunoprecipitation to study the synthesis and cleavage processing of viral proteins. J. Immunol. Methods 13:153-159. Shapiro, S. Z., M. Strand, and J. T. August. 1976. High molecular weight precursor polypeptides to structural proteins of Rauscher murine leukemia virus. J. Mol. Biol. 107:459477. Sonnabend, J. A., and R. M. Friedman. 1973. Mechanisms of interferon action, p. 210-238. In N. B. Finter (ed.), Interferon and interferon inducers. North-Holland, America, Elsevier, Amsterdam and New York. Stephenson, J. R., S. R. Tronick, and S. A. Aaronson. 1975. Murine leukemia virus mutants with temperature-sensitive defects in precursor polypeptide cleavage. Cell 6:543-548. Strand, M., and J. T. August. 1976. Structural proteins of ribonucleic acid tumor viruses: purification of envelope, core and internal components. J. Biol. Chem.
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19. Van Griensven, L. J. D. L., M. C. Baudelaire, J. Peries, R. Emanoel-Ravicovitch, and M. Boiron. 1971. On the synthesis of murine leukemia virus RNA. I. Some properties of Rauscher leukemia virus isolated from interferon-treated JLSV5 cells, p. 145-153. In Lepetit colloquia on biology and medicine, Biology of oncogenic viruses, vol. 2. North-Holland, Amsterdam. 20. Van Zaane, D., A. L. J. Gielkens, M. J. A. DekkerMichielsen, and H. P. J. Bloemers. 1975. Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus. Virology 67:544-552. 21. Vogt, V. M., and R. Eisenman. 1973. Identification of a large polypeptide precursor of avian oncornavirus proteins. Proc. Natl. Acad. Sci. U.S.A. 70:1734-1738. 22. Wright, B. S., P. A. O'Brien, G. P. Shibley, S. A. Mayyasi, and J. C. Lasfargues. 1967. Infection of an established mouse bone marrow cell line (JLS-V9) with Rauscher and Moloney murine leukemia viruses. Cancer Res. 27:1672-1677.
INFwCTION AND ImmuNrry, June 1977, p. 742-747 Copyright © 1977 American Society for Microbiology
Synthesis and Cleavage Processing of Oncornavirus Proteins During Interferon Inhibition of Virus Particle Release STUART Z. SHAPIRO, MElTE STRAND, AND ALFONS BILLIAU* Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, and Department ofHuman Biology, Rega Institute for Medical Research, University ofLeuven, Leuven, Belgium* Received for publication 24 November 1976
The effect of interferon on the rate of synthesis and the cleavage processing of viral proteins in mouse cells, chronically infected with Rauscher murine leukemia virus, has been studied by immunoprecipitation of newly synthesized viral proteins from virus-infected cells pulse-labeled with [35S]methionine. Immunoprecipitated, labeled polypeptides were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and then examined by autoradiography. Cleavage processing was studied in the same manner with cells that had been pulse-labeled and then incubated with non-radioactive media for a sufficient time to allow normal cleavage processing to occur. At a concentration that strongly inhibited the release of virus particles, interferon had no effect on the synthesis of proteins carrying antigenic determinants of the major core protein p30 or of the envelope glycoprotein gp69/71. Nor did it affect the post-translational cleavage processing of the precursors to these proteins. Similarly, interferon did not affect labeling or chasing of precursor protein carrying the p15 deterninants; labeling of p15 itself could not be studied because it does not contain methionine.
Interferon has been shown to inhibit the production of type C oncornavirus particles in chronically infected cells (1-5, 7, 19). The mechanism of this inhibition is not known. It has been postulated that interferon inhibits cytolytic virus replication by interfering with translation of viral proteins (16). However, evidence exists that this may not apply to oncornaviruses. Oncornavirus-infected cells, which undergo multiple divisions in the presence of interferon, contain unaltered or increased, rather than decreased, levels of viral proteins (7, 8), and interferon-treated cells show a larger number of membrane-associated virus particles than control cells (2-4). These findings have led to the suggestion that interferon might inhibit type C virus release instead by interfering with some late step in particle assembly or release (2, 7, 8). One limitation to this model is that as yet, only the cellular concentration of viral proteins has been measured, and no distinction has been made between the rate of synthesis of viral proteins and the accumulation of viral proteins in the cell or in cell-associated virus particles. Thus, it is possible that the rate of synthesis of the major viral proteins is decreased by interferon but the proteins are retained by the cells, thereby effectively masking the decreased rate of synthesis. The present studies were designed to directly analyze the
synthesis of specific virus proteins in interferon-treated cells and, in addition, to ascertain whether interferon treatment had any effect on the post-translational processing of the major viral proteins that occurs during virus particle assembly.
742
MATERIALS AND METHODS Cell cultures and virus. NIH/3T3 cells were a gift from A. Hackett (Cell Culture Laboratory, Naval Biomedical Research Laboratory, Oakland, Calif.). The Rauscher murine leukemia virus-infected NIH/ 3T3 cell line used in these studies was established by infection of NIH/3T3 cells with Rauscher virus propagated in a normal rat kidney cell line obtained from E. Scolnick (National Institutes of Health, Bethesda, Md.). All cells were propagated at 37°C in Dulbecco-modified Eagle medium supplemented with 10% fetal bovine serum (Flow Laboratories, Rockville, Md.), penicillin (50 ,ug/ml), streptomycin (50 ,ug/ml), neomycin (100 utg/ml), and an organic buffer system {HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 7.5 mM; MOPS [3-Nmorpholinepropanesulfonic acid], 5 mM; TES [N-
tris(hydroxymethyl)methyl-2-aminomethane-sulfonic acid], 5 mM} described by Eagle (6). Unlabeled Rauscher virus propagated in a mouse bone marrow cell line, JLSV9 (22), was provided by the John L. Smith Memorial for Cancer Research under the auspices of the Special Virus Cancer Program, National Cancer Institute. Antisera. Monospecific antisera against three purified structural proteins of Rauscher virus, gp69/
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INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION
71, p3O, and p15, was prepared as described elsewhere (16). Examination of each antiserum by the competition radioimmunoassay for reactivity toward the other viral proteins indicated no detectable antigenic cross-reactivity. Cellular proteins coprecipitated with viral proteins in the immunoprecipitation procedure are distinguished from viral specific proteins by control immunoprecipitation reactions using uninfected cells (14). Interferon. Interferon was prepared from mouse L-929 cells by infection with Newcastle disease virus as described elsewhere (5). The preparation used in the present study contained 1045 NIH reference units/ml. It contained 10% fetal bovine serum from the culture medium, so that the specific activity can be estimated to be around 103 5 reference units/mg of protein. In control experiments on JLSV5 cells, doses of 100 reference units or less had no measurable effect on cell growth. At 300 and 1,000 reference units/ml cell growth was slightly inhibited. Interferon sensitivity assay. The inherent sensitivity of cell lines to the antiviral action of interferon was tested by a vesicular stomatitis virus (VSV) yield reduction assay as described earlier (1). Briefly, cell monolayers were exposed to serial dilutions of interferon for 24 h. The cultures were then drained and infected with VSV at a multiplicity of infection of 1. After incubation for 1 h, excess virus was removed by washing, fresh medium was added, and incubation was continued for 16 h. Virus was harvested and titrated by plaque assay on L-929 cells. The sensitivity of a cell line to interferon is indicated by the concentration of interferon necessary to inhibit the VSV yield by a factor of 3 (0.5
log10).
Reverse transcriptase assay. Culture supernatants were processed for reverse transcriptase determination as follows. After clarification by low-speed centrifugation, the virus in each sample was pelleted by centrifugation at 100,000 x g for 60 min, suspended in NT buffer [100 mM NaCl, 10 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.4], sedimented again as above, and finally resuspended in 0.5 ml of NT buffer. Samples of 75 ,ul were mixed with an equal volume of 100 mM Tris-hydrochloride (pH 8.3) containing 0.4% Triton X-100; 25-Il samples of this disrupted virus were assayed in reaction mixtures containing 0. 1% Triton X-100, 50 mM Tris-hydrochloride (pH 8.3), 100 mM NaCl, 0.5 mM manganous acetate, 5 mM dithiothreitol, 1 ,uM [3H]deoxythymidine 5'-triphosphate (approximately 30,000 cpm/pmol), 1.9 AM oligodeoxythymidylic acid [oligo(dT)12-18], and 7.5 ,uM polyriboadenylic acid [poly(rA)] or 7.5 itM polydeoxyadenylic acid [poly(dA)] in a total volume of 100 ,ul. Reaction mixtures were incubated for 60 min at 35°C, 100 ,ug of yeast ribonucleic acid (RNA) was added as carrier, and the reaction was terminated by the addition of 10 volumes of TP solution (5% [wt/ wt] trichloroacetic acid and 20 mM sodium pyrophosphate). Acid-insoluble material was collected on membrane filters, washed exhaustively with TP solution and then twice with ethanol, and heatdried, and the radioactivity was counted in a toluene-based scintillant. All values were the average of
743
duplicate reactions. Each sample was also tested for the presence of inhibitory activity toward reverse transcriptase by adding 25 pl of the disrupted virus preparations to standard reaction mixtures containing purified Rauscher virus and the poly(rA) template. No inhibitory activity was found in any of these samples. The activity of the samples in reaction mixtures containing poly(rA) as template was at least 50-fold the amount of [3H]deoxythymidine 5'-triphosphate incorporated by samples in reaction mixtures containing poly(dA). Assay of virus production by [3H]uridine incorporation. Cell cultures previously treated for 24 h with interferon and parallel control cultures were incubated for 24 h with growth medium containing the appropriate concentration of interferon and [3Hluridine (39.3 Ci/mmol, New England Nuclear Corp., Boston, Mass.) at 250 ACi/75-cm2 cell culture flask (T75 flask, Falcon Plastics, Oxnard, Calif.). The supernatant fluid was harvested, clarified by low-speed centrifugation, concentrated by treatment with dry Ficoll (Pharmacia Fine Chemicals, Piscataway, N.J.), and dialyzed against 25 volumes of TEN buffer (20 mM Tris-hydrochloride, pH 7.4, 1 mM ethylenediaminetetraacetic acid, and 100 mM NaCl). The preparations were layered on top of 11ml sucrose gradients (20 to 50%, wt/wt, in TEN buffer) and centrifuged for 18 h at 30,000 rpm (SW41 rotor, Beckman Instruments, Fullerton, Calif.). The gradients were fractionated into 0.5-ml samples, and acid-precipitable radioactivity was measured. Type C oncornavirus bands at 1.16 to 1.18 g/cm3 in sucrose density gradients. Analysis of viral protein synthesis by immunoprecipitation. Cultures previously treated for 24 h with interferon (1,000 U/ml) and analogous control cultures were incubated for 2 h at 37°C with 10 ml of serum-free minimal essential medium lacking methionine. The medium was then replaced with 3 ml of the same methionine-free medium plus 250 ,uCi of [35S]-methionine per ml (about 300 Ci/mmol, Amersham-Searle, Arlington Heights, Ill.), and incubation was continued for 20 min. Cells were either lysed immediately after labeling, or the labeling medium was replaced with non-radioactive growth medium, and incubation was continued for 3 h before lysis. The concentration of interferon with which each culture had been pretreated was maintained during prelabeling, labeling, and chase incubations. Cell protein was solubilized by incubation of the cells in each T75 flask at 37°C for 15 min with 2 ml of extraction buffer, 5 mM Tris-hydrochloride (pH 9.2) containing 1% Triton X-100, 400 mM KCl, 1 mM ethylenediaminetetraacetic acid, 1 mM L-1-tosylamide phenylethylchloromethyl ketone (Sigma Chemical Co., St. Louis, Mo.), and 1 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co.). The suspension was centrifuged at 27,000 x g for 10 min. The supernatant was retained. The pellet was suspended in 1 ml of extraction buffer lacking KCl and incubated for 15 min at 37°C. This suspension was centrifuged as above, and the supernatant was collected. The pooled supernatants were twice dialyzed for 3 h against 25 volumes of TEN buffer followed by centrifugation for 1 h at 100,000 x g.
744
SHAPIRO, STRAND, AND BILLIAU
Labeled polypeptides carrying virus protein antigenic determinants were immunoprecipitated by addition of 25 gl of anti-gp69/71 serum, 30 ,ul of antip30 serum, or 50 /.l of anti-p15 serum plus 5 pg of unlabeled Rauscher virus as carrier to 1 ml of labeled cell extract, followed by incubation overnight at 40C. The precipitate was collected by centrifugation for 30 min at 4,500 x g, washed twice with TEN buffer and once with acetone, and then dissolved in electrophoresis sample buffer. Electrophoresis was performed in a high-resolution 5 to 20% gradient polyacrylamide slab gel in the presence of 0.1% sodium dodecyl sulfate as described by Maizel (11). Slab gels were dried, and autoradiography was performed with Kodak No Screen medical X-ray film (NS54T). Radiolabeled protein was measured by scanning the autoradiographs in a Joyce-Loebl microdensitometer.
INFECT. IMMUN.
tures were then challenged with the cytolytic virus VSV as outlined in Materials and Methods. Analogous cultures were washed, and incubation with the appropriate concentration of interferon was continued for 24 h, after which oncornavirus release was determined by measurement of reverse transcriptase released into the medium. Inhibition of replication of both VSV and type C virus by interferon was observed to be dose dependent (Fig. 1). Approximately 3 reference units of interferon per ml were necessary to inhibit the yield of VSV to 30% of the control value, whereas approximately 16-fold more, i.e., 50 reference U/ml, was necessary to inhibit release of particleassociated reverse transcriptase to the same extent. Similar ratios for sensitivity of VSV and type C virus were found in JLSV5 and RESULTS cells (1-5). Sensitivity of Rauscher virus-infected NIH/ MO-P Experiments for determination of oncornavi3T3 cells to interferon. A cell line (NIHI3T3) rus protein synthesis were performed with a that is relatively noninducible for endogenous great excess of interferon (i.e., 1,000 reference oncornavirus production (10) was chosen for To confirm that this dose of interthese experiments to avoid the possibility of units/ml). effectively blocked release of virus partiendogenous oncornavirus protein synthesis feron cles into the supernatant fluid, parallel culmasking an interferon effect on the synthesis of tures were incubated with 0 or 1,000 reference Rauscher virus proteins. The interferon-inof mouse interferon for 24 h, followed by duced inhibition of both the replication of a units incubation for an additional 24 h with medium cytolytic virus and of the release of Rauscher containing the same concentration of interferon virus from chronically infected cells was exam- plus [3H]uridine. The medium was then asined. sayed for reverse transcriptase activity and for Confluent monolayers of Rauscher virus-in- 3H-labeled particles. Reverse transcripfected NIH/3T3 cells were incubated for 24 h tase activityvirus from interferonwith serial dilutions of mouse interferon. Cul- treated cells in media samples (1,315 cpm) was 5% of the activity in equivalent samples from untreated Rauscher virus-infected NIH/3T3 cells (26,313 cpm). Uninfected cells showed insignificant incorpora100 tion (60 cpm). Sedimentation in a sucrose density gradient of supernatant fluid from an untreated Rauscher virus-infected culture yielded 0 3030 a peak of acid-precipitable radioactivity at 0 1.165 g/cm3 (6,544 cpm), whereas sedimentation 0 of fluid from a parallel interferon-treated culture failed to produce a distinct peak; acid0 \ precipitable radioactivity banding at 1.165 g/ w cm3 was 3,364 cpm with treated cultures and 3 5 2,200 cpm when fluids from uninfected NIH/3T3 cells were tested. It was thus concluded that Rauscher virus> I infected NIH/3T3 cells respond to interferon in much the same way as previously examined 0 1 100 1000 10 cell lines, such as JISV5 and MO-P (5), and INTERFERON (units/ml) that the dosage used in experiments measuring FIG. 1. Production ofRauscher virus and VSV in viral protein synthesis effectively reduced the Rauscher virus-infected NIH/3T3 cells exposed to in- release of type C virus particles into the superterferon for 48 h. Symbols: (-) 24- to 48-h yield of natant fluid. Effect of interferon on viral protein syntheVSV as determined by plaque assay on L-929 cells; (0) 24- to 48-h yield ofRauscher virus as determined sis and processing. Several of the structural by reverse transcritase assay. proteins of Rauscher virus have been shown to
VOL. 16, 1977
INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION
be synthesized first as higher-molecular-weight precursors, which are then cleaved during virus assembly and maturation to produce the mature virus proteins (for a review, see reference 13). The Rauscher virus envelope glycoprotein gp69/71 is the cleavage product of a glycosylated 90,000-dalton polypeptide, Pr9O. The p30 and p15 proteins of the virus are both cleavage products of the same 65,000-dalton precursor polypeptide, Pr65. The effect of interferon on the synthesis of these virus protein precursors was examined. Parallel uninfected NIH/3T3 and Rauscher virus-infected NIH/3T3 cell cultures grown to confluence were incubated for 24 h with either 0 or 1,000 reference units of mouse interferon per ml. The cultures were then washed, pulse-labeled with [35S]methionine, and processed for analysis of the synthesis of gp69/71 precursor Pr9O by immunoprecipitation with anti-gp69/71 serum (Fig. 2). After a 20-min pulse-label, more than 95% of the precipitated viral specific label was in the 90,000-dalton precursor polypeptide (Fig. 2A). Comparison ofthe amount of label in virus-specific protein peaks from treated and untreated cultures indicated that the rate of synthesis of this precursor protein was not affected by interferon treatment. A larger protein weighing 145,000 daltons, which is possibly a precursor to Pr9O, is also observable in Fig. 2A. During a 3-h chase, the 145,000-dalton polypeptide disappeared, and the concentration of Pr9O decreased as the virion envelope glycoprotein gp69/71 appeared in the cell extracts of both treated and untreated cultures (Fig. 2B). The presence of interferon had no apparent effect on the cleavage processing of the precursors to gp69/71. Analogous results demonstrating a lack of effect on protein synthesis and processing by interferon were obtained from immunoprecipitations using anti-p30 serun (Fig. 3). After a 20-min pulse-label, the predominant virus-specific protein precipitable with both sera is a 65,000-dalton precursor polypeptide (Pr65). The concentration of this precursor appeared to be the same in treated and untreated cells. A larger precursor of 76,000 daltons (Pr76) and a possible precursor weighing 145,000 daltons are also observable in Fig. 3A. Some p30 protein was already seen in a 20-min pulse-label in both interferon-treated and untreated cells. With chase, the precursor proteins were processed at similar rates, and increased amounts of virion-sized p30 protein appeared in both treated and untreated cultures (Fig. 3B). In similar experiments using an anti-p15 serum and [35S]methionine the labeling and chasing of high-molecular-weight precursor pro-
745
B
B
z
FIG. .
tig69/7
ermgp69/71
0 U)
4
30 145 90 70 MOLEULARWEIGHT x 10-3
FIG. 2. Anti-gp69/71 serum precipitates from extracts of cells pulse-labeled with [5SJmethionine. Immunoprecipitated viral specific protein synthesized in ) or absence (-----) of interferon was the presence ( resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by autoradiography. Densitometer tracings of individual lanes of the autoradiogram are depicted. (A) 35S-labeled polypeptides immunoprecipitated from extracts of Rauscher virus-infected NIHI3T3 cells pulse-labeled for 20 min. (B) 35S-labeled polypeptides immunoprecipitated from extracts of Rauscher virus-infected NIHI 3T3 cells pulse-labeled for 20 min and then chased for 3 h prior to extraction. (C) 3"S-labeled cellular polypeptides non-immunospecifically coprecipitated with unlabeled antigen immunoprecipitated from an extract of labeled, uninfected NIHI3T3 cells.
teins was also unaffected by interferon treatment. Since p15 itself does not contain methionine, the complete conversion of the precursors to p15 cannot be observed in experiments that use methionine as the labeling amino acid (15). However, lack of effect on precursor labeling and chasing makes it seem unlikely that interferon treatment affects synthesis or processing of p15.
746
SHAPIRO, STRAND, AND BILLIAU
INFECT. IMMUN.
cultures (Fig. 3B). Also, a slight increase in the amount ot labeled gp69/71 in treated cultures was noted (Fig. 2B).
l z 0 C,)
145 76 65 MOLECULAR WEIGHT
30 x
10'3
FIG. 3. Anti-p30 serum precipitates from extracts of cells pulse-labeled with [35S]methionine. Immunoprecipitates were prepared and processed as described in the legend to Fig. 2. Protein was immunoprecipitated from Rauscher virus-infected cells pulselabeled for 20 min (A) or pulse-labeled for 20 min followed by a 3-h chase prior to extraction (B). An immunoprecipitate from an extract of labeled, uninfected NIH/3T3 cells (C) is included as a control. Symbols: ( ) protein synthesized in the presence of interferon; (-----) protein synthesized in the absence of interferon.
Since the rate of synthesis of viral proteins appeared to be unaffected while virus particle release from the cell was inhibited, an accumulation of cell-associated virus proteins was expected. Indeed, such an effect on the viral p30 protein was previously detected by Friedman and Ramseur (8) using a radioimmunoprecipitation inhibition assay. In the present studies the amount of labeled p30 protein immunoprecipitated after a chase period of 3 h from interferon-treated cultures appeared to be about twice the amount precipitated from untreated
DISCUSSION These findings confirm earlier observations by Friedman and co-workers (7, 8) of continued synthesis of p30 in the presence of interferon. Moreover, the data suggest that interferon does not have any effect on the rate of p30 synthesis and that the synthesis of two other viral proteins, the envelope glycoprotein and the p15 protein, is likewise unaffected by the presence of interferon. Furthermore, the cleavage of viral protein precursors that occurs during virus particle production appeared to be unaffected by interferon, whereas at the same time particle release was effectively reduced. Several possible explanations exist for the interferon-induced inhibition of oncornavLrus production. One possibility is that the synthesis of an as yet unstudied viral protein is inhibited. Although this cannot be eliminated from consideration, the presently available data argue against it. The non-glycosylated viriorn proteins p30 and p15 are part of one large polypeptide precursor, Pr65, which also appears to contain the p12 protein (17) and may contain the only other non-glycosylated major structural protein of the virus, plO (13). Failure of interferon to affect synthesis of p30 and p15 therefore implies failure to inhibit synthesis of the precursor of all non-glycosylated major structural proteins of the virus. The synthesis of the other major structural protein, the viral envelope glycoprotein gp69/71, is also observed to be unaffected by interferon. The work of Friedman et al. (7) suggests that the synthesis of the only remaining known viral protein, reverse transcriptase, is likewise unaffected by interferon. Thus, there does not appear to be an interferon-induced inhibition of synthesis of any of the known oncornavirus structural proteins. Other possible explanations of the interferon effect on oncornaviruses include an interferoninduced block of some step in virion assembly or release. Such a mechanism could involve either virus protein or the virus RNA. The data presented here suggest that the cleavage processing of any of the three virion major structural proteins studied is the step at which such a block occurs. However, not all of the cleavages of virion proteins have been examined. Cleavages should occur in the production of the smallest-molecular-weight structural proteins, p12 and plO. Also, recent evidence suggests that the reverse transcriptase of mammalian oncornaviruses is the cleavage product of a
VOL. 16, 1977
INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION
higher-molecular-weight protein (9). These unstudied cleavage events could provide steps in virion production where interferon could act. Another, as yet unstudied question is whether the glycosylation ofthe viral envelope glycoprotein is affected by interferon treatment. When glycosylation is inhibited by 2-deoxy-i-glucose, a 70,000-dalton polypeptide is observed in place of the glycoprotein precursor Pr9O (15). An inhibition of glycosylation by interferon should likewise have been reflected by a difference in the molecular weight of the envelope protein, its glycoprotein precursor Pr9O, or both proteins, and this was not observed (Fig. 2). However, changes in sugar residues might not have been detected. Alternatively, the mechanism of interferon inhibition could be in blocking the integration of a synthesized and fully processed viral protein into budding viral particles. It should be noted that interference with cleavage, glycosylation, or integration of viral proteins would be entirely novel mechanisms of interferon action. Classically, interferon has been thought to inhibit viral production by preventing viral messenger RNA translation. It has been postulated that interferon induces the synthesis of a cell protein(s), which interferes with the ability of viral messenger RNA to combine with ribosomes (16). An interferoninduced interaction with oncornaviral RNA, preventing proper packaging into particles, might therefore be a model of interferon inhibition of oncornavirus production that more closely parallels the effect of interferon observed in other virus systems. ACKNOWLEDGMENTS This investigation was supported by Public Health Service Training Grant 5T5 GM 1674 and Grant GM 11301-11 from the National Institute of General Medical Sciences, by Public Health Service Contract 71-2251 within the virus Cancer Program of the National Cancer Institute, and by the Belgian A.S.L.K. Cancer Foundation. The technical assistance of R. Conings and Francine Cornette and the editorial help of Janine Putzeys are gratefully acknowledged. We also thank J. Thomas August for his encouragement, guidance, and assistance.
LITERATURE CITED 1. Billiau, A., V. G. Edy, E. De Clercq, H. Heremans, and P. De Somer. 1975. Influence of interferon on the synthesis of virus particles in oncornavirus carrier cell lines. III. Survey of effects on A-, B- and C-type oncornaviruses. Int. J. Cancer 15:947-953. 2. Billiau, A., V. G. Edy, H. Sobis, and P. De Somer. 1974. Influence of interferon on virus-particle synthesis in oncornavirus carrier lines. II. Evidence for a direct effect on particle release. Int. J. Cancer 14:335340. 3. Billiau, A., H. Heremans, and P. T. Allen. 1976. Trapping of oncornavirus particles at the surface of interferon-treated cells. Virology 3:537-542. 4. Billiau, A., H. Heremans, P. T. Allen, and P. De Somer. 1975. Influence of interferon on the synthesis of
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virus particles in oncornavirus carrier lines. IV. Relevance to the potential application of interferon in natural infectious diseases. J. Infect. Dis. 133(Suppl.):51-55. Billiau, A., H. Sobis, and P. De Somer. 1973. Influence of interferon on virus particle formation in different oncornavirus carrier cell lines. Int. J. Cancer 12:646653. Eagle, H. 1971. Buffer combinations for mammalian cell culture. Science 174:500-503. Friedman, R. M., E. H. Chang, J. M. Ramseur, and M. W. Myers. 1975. Interferon-directed inhibition of chronic murine leukemia virus production in cell cultures: lack of effect on intracellular viral markers. J. Virol. 16:569-574. Friedman, R. M., and J. M. Ramseur. 1974. Inhibition of murine leukemia virus production in chronically infected AKR cells: a novel effect of interferon. Proc. Natl. Acad. Sci. U.S.A. 71:3542-3544. Gerwin, B. I., S. G. Smith, and P. T. Peebles. 1975. Two active forms of RD-114 virus DNA polymerase in infected cells. Cell 6:45-52. Levy, J. A. 1973. Xenotropic viruses: murine leukemia viruses associated with NIH Swiss, NZB and other mouse strains. Science 182:1151-1153. Maizel, J. V., Jr., 1971. Polyacrylamide gel electrophoresis of viral proteins, p. 179-246. In K. Maramoroach and H. Koprowski (ed.), Methods in virology, vol. 5. Academic Press Inc., New York. Naso, R. B., L. J. Arcement, and R. B. Arlinghaus. 1975. Biosynthesis of Rauscher leukemia viral proteins. Cell 4:31-36. Shapiro, S. Z., and J. T. August. 1976. Proteolytic cleavage events in oncornavirus protein synthesis. Biochim. Biophys. Acta 458:375-396. Shapiro, S. Z., and J. T. August. 1976. The use of immunoprecipitation to study the synthesis and cleavage processing of viral proteins. J. Immunol. Methods 13:153-159. Shapiro, S. Z., M. Strand, and J. T. August. 1976. High molecular weight precursor polypeptides to structural proteins of Rauscher murine leukemia virus. J. Mol. Biol. 107:459477. Sonnabend, J. A., and R. M. Friedman. 1973. Mechanisms of interferon action, p. 210-238. In N. B. Finter (ed.), Interferon and interferon inducers. North-Holland, America, Elsevier, Amsterdam and New York. Stephenson, J. R., S. R. Tronick, and S. A. Aaronson. 1975. Murine leukemia virus mutants with temperature-sensitive defects in precursor polypeptide cleavage. Cell 6:543-548. Strand, M., and J. T. August. 1976. Structural proteins of ribonucleic acid tumor viruses: purification of envelope, core and internal components. J. Biol. Chem.
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19. Van Griensven, L. J. D. L., M. C. Baudelaire, J. Peries, R. Emanoel-Ravicovitch, and M. Boiron. 1971. On the synthesis of murine leukemia virus RNA. I. Some properties of Rauscher leukemia virus isolated from interferon-treated JLSV5 cells, p. 145-153. In Lepetit colloquia on biology and medicine, Biology of oncogenic viruses, vol. 2. North-Holland, Amsterdam. 20. Van Zaane, D., A. L. J. Gielkens, M. J. A. DekkerMichielsen, and H. P. J. Bloemers. 1975. Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus. Virology 67:544-552. 21. Vogt, V. M., and R. Eisenman. 1973. Identification of a large polypeptide precursor of avian oncornavirus proteins. Proc. Natl. Acad. Sci. U.S.A. 70:1734-1738. 22. Wright, B. S., P. A. O'Brien, G. P. Shibley, S. A. Mayyasi, and J. C. Lasfargues. 1967. Infection of an established mouse bone marrow cell line (JLS-V9) with Rauscher and Moloney murine leukemia viruses. Cancer Res. 27:1672-1677.
