INFwCTION AND ImmuNrry, June 1977, p. 742-747Copyright © 1977 American Society for Microbiology

Vol. 16, No. 3Printed in U.S.A.

Synthesis and Cleavage Processing of Oncornavirus ProteinsDuring 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, andDepartment 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 ofviral proteins in mouse cells, chronically infected with Rauscher murine leuke-mia virus, has been studied by immunoprecipitation of newly synthesized viralproteins from virus-infected cells pulse-labeled with [35S]methionine. Immuno-precipitated, labeled polypeptides were resolved by polyacrylamide gel electro-phoresis in the presence of sodium dodecyl sulfate and then examined byautoradiography. Cleavage processing was studied in the same manner withcells that had been pulse-labeled and then incubated with non-radioactive mediafor a sufficient time to allow normal cleavage processing to occur. At a concen-tration that strongly inhibited the release of virus particles, interferon had noeffect on the synthesis of proteins carrying antigenic determinants of the majorcore protein p30 or of the envelope glycoprotein gp69/71. Nor did it affect thepost-translational cleavage processing of the precursors to these proteins. Simi-larly, interferon did not affect labeling or chasing of precursor protein carryingthe p15 deterninants; labeling of p15 itself could not be studied because it doesnot contain methionine.

Interferon has been shown to inhibit the pro-duction of type C oncornavirus particles inchronically infected cells (1-5, 7, 19). The mech-anism of this inhibition is not known. It hasbeen postulated that interferon inhibits cytoly-tic virus replication by interfering with transla-tion of viral proteins (16). However, evidenceexists that this may not apply to oncornavi-ruses. Oncornavirus-infected cells, which un-dergo multiple divisions in the presence of in-terferon, contain unaltered or increased, ratherthan decreased, levels of viral proteins (7, 8),and interferon-treated cells show a larger num-ber of membrane-associated virus particlesthan control cells (2-4). These findings have ledto the suggestion that interferon might inhibittype C virus release instead by interfering withsome late step in particle assembly or release(2, 7, 8). One limitation to this model is that asyet, only the cellular concentration of viral pro-teins has been measured, and no distinctionhas been made between the rate of synthesis ofviral proteins and the accumulation of viralproteins in the cell or in cell-associated virusparticles. Thus, it is possible that the rate ofsynthesis of the major viral proteins is de-creased by interferon but the proteins are re-tained by the cells, thereby effectively maskingthe decreased rate of synthesis. The presentstudies were designed to directly analyze the

synthesis of specific virus proteins in inter-feron-treated cells and, in addition, to ascertainwhether interferon treatment had any effect onthe post-translational processing of the majorviral proteins that occurs during virus particleassembly.

MATERIALS AND METHODSCell cultures and virus. NIH/3T3 cells were a gift

from A. Hackett (Cell Culture Laboratory, NavalBiomedical Research Laboratory, Oakland, Calif.).The Rauscher murine leukemia virus-infected NIH/3T3 cell line used in these studies was establishedby infection of NIH/3T3 cells with Rauscher viruspropagated in a normal rat kidney cell line obtainedfrom E. Scolnick (National Institutes of Health,Bethesda, Md.). All cells were propagated at 37°Cin Dulbecco-modified Eagle medium supplementedwith 10% fetal bovine serum (Flow Laboratories,Rockville, Md.), penicillin (50 ,ug/ml), streptomycin(50 ,ug/ml), neomycin (100 utg/ml), and an organicbuffer system {HEPES [N-2-hydroxyethylpipera-zine-N'-2-ethanesulfonic acid], 7.5 mM; MOPS [3-N-morpholinepropanesulfonic acid], 5 mM; TES [N-tris(hydroxymethyl)methyl-2-aminomethane-sul-fonic acid], 5 mM} described by Eagle (6). Un-labeled Rauscher virus propagated in a mouse bonemarrow cell line, JLSV9 (22), was provided by theJohn L. Smith Memorial for Cancer Research underthe auspices of the Special Virus Cancer Program,National Cancer Institute.

Antisera. Monospecific antisera against threepurified structural proteins of Rauscher virus, gp69/

742

on August 11, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION 743

71, p3O, and p15, was prepared as described else-where (16). Examination of each antiserum by thecompetition radioimmunoassay for reactivity to-ward the other viral proteins indicated no detectableantigenic cross-reactivity. Cellular proteins copre-cipitated with viral proteins in the immunoprecipi-tation procedure are distinguished from viral spe-cific proteins by control immunoprecipitation reac-tions using uninfected cells (14).

Interferon. Interferon was prepared from mouseL-929 cells by infection with Newcastle disease virusas described elsewhere (5). The preparation used inthe present study contained 1045 NIH referenceunits/ml. It contained 10% fetal bovine serum fromthe culture medium, so that the specific activity canbe estimated to be around 103 5 reference units/mg ofprotein. In control experiments on JLSV5 cells,doses of 100 reference units or less had no measura-ble effect on cell growth. At 300 and 1,000 referenceunits/ml cell growth was slightly inhibited.

Interferon sensitivity assay. The inherent sensi-tivity of cell lines to the antiviral action of inter-feron was tested by a vesicular stomatitis virus(VSV) yield reduction assay as described earlier (1).Briefly, cell monolayers were exposed to serial dilu-tions of interferon for 24 h. The cultures were thendrained and infected with VSV at a multiplicity ofinfection of 1. After incubation for 1 h, excess viruswas removed by washing, fresh medium was added,and incubation was continued for 16 h. Virus washarvested and titrated by plaque assay on L-929cells. The sensitivity of a cell line to interferon isindicated by the concentration of interferon neces-sary to inhibit the VSV yield by a factor of 3 (0.5log10).

Reverse transcriptase assay. Culture superna-tants were processed for reverse transcriptase deter-mination as follows. After clarification by low-speedcentrifugation, the virus in each sample was pel-leted by centrifugation at 100,000 x g for 60 min,suspended in NT buffer [100 mM NaCl, 10 mMtris(hydroxymethyl)aminomethane (Tris)-hydro-chloride, pH 7.4], sedimented again as above, andfinally resuspended in 0.5 ml ofNT buffer. Samplesof 75 ,ul were mixed with an equal volume of 100mMTris-hydrochloride (pH 8.3) containing 0.4% TritonX-100; 25-Il samples of this disrupted virus wereassayed in reaction mixtures containing 0.1% TritonX-100, 50 mM Tris-hydrochloride (pH 8.3), 100 mMNaCl, 0.5 mM manganous acetate, 5 mM dithiothrei-tol, 1 ,uM [3H]deoxythymidine 5'-triphosphate (ap-proximately 30,000 cpm/pmol), 1.9 AM oligodeoxy-thymidylic acid [oligo(dT)12-18], and 7.5 ,uM polyri-boadenylic acid [poly(rA)] or 7.5 itM polydeoxyaden-ylic acid [poly(dA)] in a total volume of 100 ,ul.Reaction mixtures were incubated for 60 min at35°C, 100 ,ug of yeast ribonucleic acid (RNA) wasadded as carrier, and the reaction was terminatedby the addition of 10 volumes ofTP solution (5% [wt/wt] trichloroacetic acid and 20 mM sodium pyro-phosphate). Acid-insoluble material was collectedon membrane filters, washed exhaustively with TPsolution and then twice with ethanol, and heat-dried, and the radioactivity was counted in a tolu-ene-based scintillant. All values were the average of

duplicate reactions. Each sample was also tested forthe presence of inhibitory activity toward reversetranscriptase by adding 25 pl of the disrupted viruspreparations to standard reaction mixtures contain-ing purified Rauscher virus and the poly(rA) tem-plate. No inhibitory activity was found in any ofthese samples. The activity of the samples in reac-tion mixtures containing poly(rA) as template wasat least 50-fold the amount of [3H]deoxythymidine5'-triphosphate incorporated by samples in reactionmixtures containing poly(dA).

Assay of virus production by [3H]uridine incorpo-ration. Cell cultures previously treated for 24 h withinterferon and parallel control cultures were incu-bated for 24 h with growth medium containing theappropriate concentration of interferon and[3Hluridine (39.3 Ci/mmol, New England NuclearCorp., Boston, Mass.) at 250 ACi/75-cm2 cell cultureflask (T75 flask, Falcon Plastics, Oxnard, Calif.).The supernatant fluid was harvested, clarified bylow-speed centrifugation, concentrated by treat-ment with dry Ficoll (Pharmacia Fine Chemicals,Piscataway, N.J.), and dialyzed against 25 volumesofTEN buffer (20 mM Tris-hydrochloride, pH 7.4, 1mM ethylenediaminetetraacetic acid, and 100 mMNaCl). The preparations were layered on top of 11-ml sucrose gradients (20 to 50%, wt/wt, in TENbuffer) and centrifuged for 18 h at 30,000 rpm (SW41rotor, Beckman Instruments, Fullerton, Calif.). Thegradients 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 insucrose density gradients.

Analysis of viral protein synthesis by immuno-precipitation. Cultures previously treated for 24 hwith interferon (1,000 U/ml) and analogous controlcultures were incubated for 2 h at 37°C with 10 ml ofserum-free minimal essential medium lacking me-thionine. The medium was then replaced with 3 mlof the same methionine-free medium plus 250 ,uCi of[35S]-methionine per ml (about 300 Ci/mmol, Amer-sham-Searle, Arlington Heights, Ill.), and incuba-tion was continued for 20 min. Cells were eitherlysed immediately after labeling, or the labeling me-dium was replaced with non-radioactive growth me-dium, and incubation was continued for 3 h beforelysis. The concentration of interferon with whicheach culture had been pretreated was maintainedduring prelabeling, labeling, and chase incubations.Cell protein was solubilized by incubation of thecells in each T75 flask at 37°C for 15 min with 2 ml ofextraction buffer, 5 mM Tris-hydrochloride (pH 9.2)containing 1% Triton X-100, 400 mM KCl, 1 mMethylenediaminetetraacetic acid, 1 mM L-1-tosyla-mide phenylethylchloromethyl ketone (Sigma Chem-ical Co., St. Louis, Mo.), and 1 mM phenylmethyl-sulfonyl fluoride (Sigma Chemical Co.). The suspen-sion was centrifuged at 27,000 x g for 10 min. Thesupernatant was retained. The pellet was suspendedin 1 ml of extraction buffer lacking KCl and incu-bated for 15 min at 37°C. This suspension was centri-fuged as above, and the supernatant was collected.The pooled supernatants were twice dialyzed for 3 hagainst 25 volumes of TEN buffer followed by cen-trifugation for 1 h at 100,000 x g.

VOL. 16, 1977

on August 11, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

744 SHAPIRO, STRAND, AND BILLIAU

Labeled polypeptides carrying virus protein anti-genic determinants were immunoprecipitated by ad-dition of 25 gl of anti-gp69/71 serum, 30 ,ul of anti-p30 serum, or 50 /.l of anti-p15 serum plus 5 pg ofunlabeled Rauscher virus as carrier to 1 ml of la-beled cell extract, followed by incubation overnightat 40C. The precipitate was collected by centrifuga-tion for 30 min at 4,500 x g, washed twice with TENbuffer and once with acetone, and then dissolved inelectrophoresis sample buffer. Electrophoresis wasperformed in a high-resolution 5 to 20% gradientpolyacrylamide slab gel in the presence of 0.1% so-dium dodecyl sulfate as described by Maizel (11).Slab gels were dried, and autoradiography was per-formed with Kodak No Screen medical X-ray film(NS54T). Radiolabeled protein was measured byscanning the autoradiographs in a Joyce-Loebl mi-crodensitometer.

RESULTSSensitivity of Rauscher virus-infected NIH/

3T3 cells to interferon. A cell line (NIHI3T3)that is relatively noninducible for endogenousoncornavirus production (10) was chosen forthese experiments to avoid the possibility ofendogenous oncornavirus protein synthesismasking an interferon effect on the synthesis ofRauscher virus proteins. The interferon-in-duced inhibition of both the replication of acytolytic virus and of the release of Rauschervirus from chronically infected cells was exam-ined.

Confluent monolayers of Rauscher virus-in-fected NIH/3T3 cells were incubated for 24 hwith serial dilutions of mouse interferon. Cul-

100

0 30300

0

0 \w5 3

> I

0 1 10 100 1000INTERFERON (units/ml)

FIG. 1. Production ofRauscher virus and VSV inRauscher virus-infected NIH/3T3 cells exposed to in-terferon for 48 h. Symbols: (-) 24- to 48-h yield ofVSV as determined by plaque assay on L-929 cells;(0) 24- to 48-h yield ofRauscher virus as determinedby reverse transcritase assay.

tures were then challenged with the cytolyticvirus VSV as outlined in Materials and Meth-ods. Analogous cultures were washed, and in-cubation with the appropriate concentration ofinterferon was continued for 24 h, after whichoncornavirus release was determined by mea-surement of reverse transcriptase released intothe medium. Inhibition of replication of bothVSV and type C virus by interferon was ob-served to be dose dependent (Fig. 1). Approxi-mately 3 reference units of interferon per mlwere necessary to inhibit the yield of VSV to30% of the control value, whereas approxi-mately 16-fold more, i.e., 50 reference U/ml,was necessary to inhibit release of particle-associated reverse transcriptase to the sameextent. Similar ratios for sensitivity of VSVand type C virus were found in JLSV5 andMO-P cells (1-5).Experiments for determination of oncornavi-

rus protein synthesis were performed with agreat excess of interferon (i.e., 1,000 referenceunits/ml). To confirm that this dose of inter-feron effectively blocked release of virus parti-cles into the supernatant fluid, parallel cul-tures were incubated with 0 or 1,000 referenceunits of mouse interferon for 24 h, followed byincubation for an additional 24 h with mediumcontaining the same concentration ofinterferonplus [3H]uridine. The medium was then as-sayed for reverse transcriptase activity and for3H-labeled virus particles. Reverse transcrip-tase activity in media samples from interferon-treated cells (1,315 cpm) was 5% of the activityin equivalent samples from untreated Rauschervirus-infected NIH/3T3 cells (26,313 cpm). Un-infected cells showed insignificant incorpora-tion (60 cpm). Sedimentation in a sucrose den-sity gradient of supernatant fluid from an un-treated Rauscher virus-infected culture yieldeda peak of acid-precipitable radioactivity at1.165 g/cm3 (6,544 cpm), whereas sedimentationof fluid from a parallel interferon-treated cul-ture failed to produce a distinct peak; acid-precipitable radioactivity banding at 1.165 g/cm3 was 3,364 cpm with treated cultures and2,200 cpm when fluids from uninfected NIH/3T3cells were tested.

It was thus concluded that Rauscher virus-infected NIH/3T3 cells respond to interferon inmuch the same way as previously examinedcell lines, such as JISV5 and MO-P (5), andthat the dosage used in experiments measuringviral protein synthesis effectively reduced therelease of type C virus particles into the super-natant fluid.

Effect of interferon on viral protein synthe-sis and processing. Several of the structuralproteins of Rauscher virus have been shown to

INFECT. IMMUN.

on August 11, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION 745

be synthesized first as higher-molecular-weightprecursors, which are then cleaved during virusassembly and maturation to produce the ma-ture virus proteins (for a review, see reference13). The Rauscher virus envelope glycoproteingp69/71 is the cleavage product of a glycosyl-ated 90,000-dalton polypeptide, Pr9O. The p30and p15 proteins of the virus are both cleavageproducts of the same 65,000-dalton precursorpolypeptide, Pr65. The effect of interferon onthe synthesis of these virus protein precursorswas examined.

Parallel uninfected NIH/3T3 and Rauschervirus-infected NIH/3T3 cell cultures grown toconfluence were incubated for 24 h with either 0or 1,000 reference units of mouse interferon perml. The cultures were then washed, pulse-la-beled with [35S]methionine, and processed foranalysis of the synthesis of gp69/71 precursorPr9O by immunoprecipitation with anti-gp69/71serum (Fig. 2). After a 20-min pulse-label, morethan 95% of the precipitated viral specific labelwas in the 90,000-dalton precursor polypeptide(Fig. 2A). Comparison ofthe amount of label invirus-specific protein peaks from treated anduntreated cultures indicated that the rate ofsynthesis of this precursor protein was not af-fected by interferon treatment. A larger proteinweighing 145,000 daltons, which is possibly aprecursor to Pr9O, is also observable in Fig. 2A.During a 3-h chase, the 145,000-dalton polypep-tide disappeared, and the concentration of Pr9Odecreased as the virion envelope glycoproteingp69/71 appeared in the cell extracts of bothtreated and untreated cultures (Fig. 2B). Thepresence of interferon had no apparent effect onthe cleavage processing of the precursors togp69/71.Analogous results demonstrating a lack of

effect on protein synthesis and processing byinterferon were obtained from immunoprecipi-tations using anti-p30 serun (Fig. 3). After a20-min pulse-label, the predominant virus-spe-cific protein precipitable with both sera is a65,000-dalton precursor polypeptide (Pr65). Theconcentration of this precursor appeared to bethe same in treated and untreated cells. Alarger precursor of 76,000 daltons (Pr76) and apossible precursor weighing 145,000 daltons arealso observable in Fig. 3A. Some p30 proteinwas already seen in a 20-min pulse-label inboth interferon-treated and untreated cells.With chase, the precursor proteins were proc-essed at similar rates, and increased amountsof virion-sized p30 protein appeared in bothtreated and untreated cultures (Fig. 3B).

In similar experiments using an anti-p15 se-rum and [35S]methionine the labeling and chas-ing of high-molecular-weight precursor pro-

B

BzFIG . . tig69/7 ermgp69/71

0U)

4

14590 70 30MOLEULARWEIGHT x 10-3

FIG. 2. Anti-gp69/71 serum precipitates from ex-tracts ofcells pulse-labeled with [5SJmethionine. Im-munoprecipitated viral specific protein synthesized inthe presence ( ) or absence (-----) of interferon wasresolved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis and examined by autoradiogra-phy. Densitometer tracings of individual lanes of theautoradiogram are depicted. (A) 35S-labeled polypep-tides immunoprecipitated from extracts of Rauschervirus-infected NIHI3T3 cells pulse-labeled for 20min. (B) 35S-labeled polypeptides immunoprecipi-tated from extracts of Rauscher virus-infected NIHI3T3 cells pulse-labeled for 20 min and then chasedfor 3 h prior to extraction. (C) 3"S-labeled cellularpolypeptides non-immunospecifically coprecipitatedwith unlabeled antigen immunoprecipitated from anextract of labeled, uninfected NIHI3T3 cells.

teins was also unaffected by interferon treat-ment. Since p15 itself does not contain methio-nine, the complete conversion of the precursorsto p15 cannot be observed in experiments thatuse methionine as the labeling amino acid (15).However, lack of effect on precursor labelingand chasing makes it seem unlikely that inter-feron treatment affects synthesis or processingof p15.

VOL. 16, 1977

on August 11, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

746 SHAPIRO, STRAND, AND BILLIAU

z

0C,)

145 76 65 30MOLECULAR WEIGHT x 10'3

FIG. 3. Anti-p30 serum precipitates from extractsofcells pulse-labeled with [35S]methionine. Immuno-precipitates were prepared and processed as de-scribed in the legend to Fig. 2. Protein was immuno-precipitated from Rauscher virus-infected cells pulse-labeled for 20 min (A) or pulse-labeled for 20 minfollowed by a 3-h chase prior to extraction (B). Animmunoprecipitate from an extract of labeled, unin-fected NIH/3T3 cells (C) is included as a control.Symbols: ( ) protein synthesized in the presence ofinterferon; (-----) protein synthesized in the absenceof interferon.

Since the rate of synthesis of viral proteinsappeared to be unaffected while virus particlerelease from the cell was inhibited, an accumu-lation of cell-associated virus proteins was ex-

pected. Indeed, such an effect on the viral p30protein was previously detected by Friedmanand Ramseur (8) using a radioimmunoprecipi-tation inhibition assay. In the present studiesthe amount of labeled p30 protein immunopre-cipitated after a chase period of 3 h from inter-feron-treated cultures appeared to be abouttwice the amount precipitated from untreated

cultures (Fig. 3B). Also, a slight increase in theamount ot labeled gp69/71 in treated cultureswas noted (Fig. 2B).

DISCUSSIONThese findings confirm earlier observations

by Friedman and co-workers (7, 8) of continuedsynthesis of p30 in the presence of interferon.Moreover, the data suggest that interferon doesnot have any effect on the rate of p30 synthesisand that the synthesis of two other viral pro-teins, the envelope glycoprotein and the p15protein, is likewise unaffected by the presenceof interferon. Furthermore, the cleavage ofviral protein precursors that occurs during vi-rus particle production appeared to be unaf-fected by interferon, whereas at the same timeparticle release was effectively reduced.

Several possible explanations exist for theinterferon-induced inhibition of oncornavLrusproduction. One possibility is that the synthesisofan as yet unstudied viral protein is inhibited.Although this cannot be eliminated from con-sideration, the presently available data argueagainst it. The non-glycosylated viriorn proteinsp30 and p15 are part of one large polypeptideprecursor, Pr65, which also appears to containthe p12 protein (17) and may contain the onlyother non-glycosylated major structural proteinof the virus, plO (13). Failure of interferon toaffect synthesis ofp30 and p15 therefore impliesfailure to inhibit synthesis of the precursor ofall non-glycosylated major structural proteinsof the virus. The synthesis of the other majorstructural protein, the viral envelope glycopro-tein gp69/71, is also observed to be unaffectedby interferon. The work of Friedman et al. (7)suggests that the synthesis of the only remain-ing known viral protein, reverse transcriptase,is likewise unaffected by interferon. Thus,there does not appear to be an interferon-in-duced inhibition of synthesis of any of theknown oncornavirus structural proteins.Other possible explanations of the interferon

effect on oncornaviruses include an interferon-induced block of some step in virion assemblyor release. Such a mechanism could involveeither virus protein or the virus RNA. The datapresented here suggest that the cleavage proc-essing of any of the three virion major struc-tural proteins studied is the step at which sucha block occurs. However, not all of the cleav-ages of virion proteins have been examined.Cleavages should occur in the production of thesmallest-molecular-weight structural proteins,p12 and plO. Also, recent evidence suggeststhat the reverse transcriptase of mammalianoncornaviruses is the cleavage product of a

l

INFECT. IMMUN.

on August 11, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

INTERFERON AND ONCORNAVIRUS PROTEIN PRODUCTION 747

higher-molecular-weight protein (9). These un-studied cleavage events could provide steps invirion production where interferon could act.Another, as yet unstudied question is whetherthe glycosylation ofthe viral envelope glycopro-tein is affected by interferon treatment. Whenglycosylation is inhibited by 2-deoxy-i-glucose,a 70,000-dalton polypeptide is observed in placeof the glycoprotein precursor Pr9O (15). An inhi-bition of glycosylation by interferon shouldlikewise have been reflected by a difference inthe molecular weight of the envelope protein,its glycoprotein precursor Pr9O, or both pro-teins, and this was not observed (Fig. 2). How-ever, changes in sugar residues might not havebeen detected. Alternatively, the mechanism ofinterferon inhibition could be in blocking theintegration ofa synthesized and fully processedviral protein into budding viral particles. Itshould be noted that interference with cleav-age, glycosylation, or integration of viral pro-teins would be entirely novel mechanisms ofinterferon action. Classically, interferon hasbeen thought to inhibit viral production by pre-venting viral messenger RNA translation. Ithas been postulated that interferon induces thesynthesis of a cell protein(s), which interfereswith the ability of viral messenger RNA tocombine with ribosomes (16). An interferon-induced interaction with oncornaviral RNA,preventing proper packaging into particles,might therefore be a model of interferon inhibi-tion of oncornavirus production that moreclosely parallels the effect of interferon ob-served in other virus systems.

ACKNOWLEDGMENTSThis investigation was supported by Public Health Ser-

vice Training Grant 5T5 GM 1674 and Grant GM 11301-11from the National Institute of General Medical Sciences, byPublic Health Service Contract 71-2251 within the virusCancer Program of the National Cancer Institute, and bythe Belgian A.S.L.K. Cancer Foundation. The technicalassistance of R. Conings and Francine Cornette and theeditorial help of Janine Putzeys are gratefully acknowl-edged. We also thank J. Thomas August for his encourage-ment, guidance, and assistance.

LITERATURE CITED1. Billiau, A., V. G. Edy, E. De Clercq, H. Heremans, and

P. De Somer. 1975. Influence of interferon on thesynthesis of virus particles in oncornavirus carriercell lines. III. Survey of effects on A-, B- and C-typeoncornaviruses. Int. J. Cancer 15:947-953.

2. Billiau, A., V. G. Edy, H. Sobis, and P. De Somer.1974. Influence ofinterferon on virus-particle synthe-sis in oncornavirus carrier lines. II. Evidence for adirect effect on particle release. Int. J. Cancer 14:335-340.

3. Billiau, A., H. Heremans, and P. T. Allen. 1976. Trap-ping of oncornavirus particles at the surface of inter-feron-treated cells. Virology 3:537-542.

4. Billiau, A., H. Heremans, P. T. Allen, and P. De So-mer. 1975. Influence of interferon on the synthesis of

virus particles in oncornavirus carrier lines. IV. Rele-vance to the potential application of interferon innatural infectious diseases. J. Infect. Dis.133(Suppl.):51-55.

5. Billiau, A., H. Sobis, and P. De Somer. 1973. Influenceof interferon on virus particle formation in differentoncornavirus carrier cell lines. Int. J. Cancer 12:646-653.

6. Eagle, H. 1971. Buffer combinations for mammaliancell culture. Science 174:500-503.

7. Friedman, R. M., E. H. Chang, J. M. Ramseur, and M.W. Myers. 1975. Interferon-directed inhibition ofchronic murine leukemia virus production in cell cul-tures: lack of effect on intracellular viral markers. J.Virol. 16:569-574.

8. Friedman, R. M., and J. M. Ramseur. 1974. Inhibitionof murine leukemia virus production in chronicallyinfected AKR cells: a novel effect of interferon. Proc.Natl. Acad. Sci. U.S.A. 71:3542-3544.

9. Gerwin, B. I., S. G. Smith, and P. T. Peebles. 1975.Two active forms of RD-114 virus DNA polymerase ininfected cells. Cell 6:45-52.

10. Levy, J. A. 1973. Xenotropic viruses: murine leukemiaviruses associated with NIH Swiss, NZB and othermouse strains. Science 182:1151-1153.

11. Maizel, J. V., Jr., 1971. Polyacrylamide gel electropho-resis of viral proteins, p. 179-246. In K. Maramoroachand H. Koprowski (ed.), Methods in virology, vol. 5.Academic Press Inc., New York.

12. Naso, R. B., L. J. Arcement, and R. B. Arlinghaus.1975. Biosynthesis of Rauscher leukemia viral pro-teins. Cell 4:31-36.

13. Shapiro, S. Z., and J. T. August. 1976. Proteolyticcleavage events in oncornavirus protein synthesis.Biochim. Biophys. Acta 458:375-396.

14. Shapiro, S. Z., and J. T. August. 1976. The use ofimmunoprecipitation to study the synthesis andcleavage processing of viral proteins. J. Immunol.Methods 13:153-159.

15. Shapiro, S. Z., M. Strand, and J. T. August. 1976. Highmolecular weight precursor polypeptides to struc-tural proteins of Rauscher murine leukemia virus. J.Mol. Biol. 107:459477.

16. Sonnabend, J. A., and R. M. Friedman. 1973. Mecha-nisms of interferon action, p. 210-238. In N. B. Finter(ed.), Interferon and interferon inducers. North-Hol-land, America, Elsevier, Amsterdam and New York.

17. Stephenson, J. R., S. R. Tronick, and S. A. Aaronson.1975. Murine leukemia virus mutants with tempera-ture-sensitive defects in precursor polypeptide cleav-age. Cell 6:543-548.

18. Strand, M., and J. T. August. 1976. Structural proteinsof ribonucleic acid tumor viruses: purification of en-velope, core and internal components. J. Biol. Chem.251:559-564.

19. Van Griensven, L. J. D. L., M. C. Baudelaire, J. Peries,R. Emanoel-Ravicovitch, and M. Boiron. 1971. Onthe synthesis ofmurine leukemia virus RNA. I. Someproperties of Rauscher leukemia virus isolated frominterferon-treated JLSV5 cells, p. 145-153. In Lepetitcolloquia on biology and medicine, Biology of onco-genic viruses, vol. 2. North-Holland, Amsterdam.

20. Van Zaane, D., A. L. J. Gielkens, M. J. A. Dekker-Michielsen, and H. P. J. Bloemers. 1975. Virus-spe-cific precursor polypeptides in cells infected withRauscher leukemia virus. Virology 67:544-552.

21. Vogt, V. M., and R. Eisenman. 1973. Identification ofalarge polypeptide precursor of avian oncornavirusproteins. 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 anestablished mouse bone marrow cell line (JLS-V9)with Rauscher and Moloney murine leukemia vi-ruses. Cancer Res. 27:1672-1677.

VOL. 16, 1977

on August 11, 2018 by guest

http://iai.asm.org/

Dow

nloaded from