Vol. 41, No. 3 JOURNALOFVIROLOGY, Mar. 1982, p. 1109-1111
0022-538X/82/031109-03$02.00/0
Effect of the Arginine Analog Canavanine on the Synthesis of Epstein-Barr Virus-Induced Proteins in Superinfected Raji
Cells
GARY J. BAYLISS* AND HANS WOLF
MaxvonPettenkofer Institute, 8000 Munich 2, Federal Republic of Germany Received 10 August1981/Accepted 27 October 1981
Theaddition ofcanavanine to cultures of superinfected Rajicellsin theabsence ofarginine preventedthe appearanceofearlyantigensasdefined byimmunofluo- rescentstaining. Addition of variousamountsof argininepermitted the identifica- tion ofatleastthree groupsofproteins, eachresponding differently to the various concentrations of arginine-canavanine.
Superinfection of Raji cells withEpstein-Barr virus (EBV) derived from P3HR1 cells (HR1- EBV) leadstothesynthesisofavariety of virus- induced proteins which can be divided into several groups according to their kinetics of synthesis, theirrequirement for viralDNA syn- thesisresponse tocycloheximide blocksapplied atdifferent times afterinfection, and their syn- thesis in thepresence of amino acid analogs (1, la, 2, 4-6, 8). Previous studies byus indicated that treatment ofsuperinfected Raji cells with canavanine in the absenceofarginine prevented thesynthesis ofEBVearly antigens (EA). Only a minimal expression of the virus could be observed; only three virus-inducedpolypeptides (120, 102, and 49)were identified inextractsof suchcells. Treatment with azetidine (ananalog ofproline) didnothavethis drastic effect since the cells proceeded to EA synthesis but not viruscapsid antigen (VCA) synthesis. A recent report (9) suggested that canavanine did not suppress the synthesis of EA in superinfected Raji cells.
Since the control of synthesis of early or intermediate earlyproteins in cells infected with oncogenic viruses can be seen as one of the factors that will determine whether the cell will become transformed or enter alytic cycle, we investigated thisapparentdiscrepancy. Firstwe asked the question why canavanine has such a strong effect whereas azetidine is much less effective. Sinceitwas shown that the synthesis of certainherpessimplexvirusproteinscouldbe affected by varying the arginine content ofthe culture medium (3, 7), we investigated the be- havior of superinfected Raji cells under condi- tionsofarginine starvation.
Raji cells were superinfected using a virus stock that had been diluted with arginine-free medium. After1 hofadsorption the cellswere washed with arginine-free medium twice and
resuspended in either arginine-free medium or medium containing the normal amount of argi- nine. After 12 h the cells were labeled with [32S]methionine in arginine-free or -containing medium. Figure1Billustrates the resultsof such anexperiment; S tracks aresuperinfected cells, andtracksindicated witha-aremock-infected controls. Therewas aslight overall reduction in protein synthesis in the absenceofarginine, but allof the virus proteins could be identified. In a parallelset of cultures weincluded canavanine at a concentration of 0.9 mM, with arginine present at 0, 1, 10, or 100% of its normal concentration (0.95 mM) (Fig. IA). As can be seen from this experiment, the EBV-induced proteinsfall intoatleast threegroups: (i) those synthesized in thepresenceof0.9 mM canavan- ine regardless of the arginine concentration (e.g., protein 138); (ii) those proteins synthe- sized in the presence of 0.9 mM canavanine when arginine is present at 10 or 100% of its normal concentration (e.g.,protein 150); and (iii) thoseproteins synthesized only whencanavan- ine is omitted from the culture medium (e.g., protein 143).
Inthis experiment all superinfected cultures werepositivefor EBV EA. Ourprevious study (la) suggested that EA was not synthesized in the presence of 2.7 mM canavanine in the ab- sence of arginine. To confirm this finding we superinfected Raji cells in the absence ofargi- nine and then incubated the cells for 12 h in media containing 0, 0.1, 0.3, 0.9, or 2.7 mM canavanineand0, 1, 10, or100% ofthenormal arginine concentration.After 12 h the cellswere stained for EA and processed for analysis on polyacrylamide gels(Fig. 1D)orforimmunopre- cipitation (Fig. 1C). When superinfected Raji cellswere incubatedin thepresence of2.7 mM canavanine in mediawith0 or1% ofthe normal arginine concentration they remained negative 1109
A
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0
B
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'm-
49
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FIG. 1. Polyacrylamide gel analysisof EBV-inducedproteins synthesizedinthepresenceofvaryingamounts of canavanine andarginine.The virus-inducedproteinsarelabeled with theirapproximate molecularweights.
Protein90/Hisacomplexbandcontainingboth virus and hostproteins. (AandB) Rajicellsweresuperinfected (S)ormock-infected(-)with HR1-EBV in thepresenceof0.9 mM canavanine(A)orthe absence of canavanine (B). Argininewaspresentat0, 1, 10,or100% of its normal concentration(tracks 0, 1, 10,andN, respectively).
After adsorption the cultures were incubated for12 h in media with the relevant arginine and canavanine concentrations. The cellswerethenpulse-labeled (30min)with[35S]methionine (50 p.Ci/ml)in methionine-free medium containing the relevant concentrations of canavanine and arginine. The cells were disrupted in electrophoresis sample buffer andanalyzedon10%opolyacrylamide gelscross-linkedwithdiallyltartardiamide containing2% sodiumdodecylsulfate(formethoddetails,seereferencela). (C) Rajicellsweresuperinfectedin thepresence(+C) orabsence of 2.7 mMcanavanine; argininewas either absent(0) orpresentat its normal concentration(N). The cells wereincubated for 12 h, labeled asdescribed for(A) and (B) for 4 h and then preparedforimmunoprecipitation (as described in reference la). Theimmunoprecipitates were analyzedon polyacrylamide gelsasdescribed.(D) Rajicellsweresuperinfectedinthepresenceof canavanine(0, 0.1, 0.3, 0.7, and 2.7mM)and various concentrationsofarginine (0, 1,10or100% of its normalconcentration). At12 h after infection the cellswereprocessedasdescribed for(A)and(B).
15 ISO 143 138 110
9#0 so
63 se
49 45 4340
NOTES 1111 for EA; all other cultures were positive for this
antigencomplex. The immune precipitation re- vealed that at0% arginineand 2.7 mMcanavan- ine no immune precipitable proteins were syn- thesized(Fig. 1C, track0 + C), whereas those cultures positive forEAcontained avariety of immune precipitable EBV-induced proteins (Fig. 1C, tracks N, N + C, and 0). The treated anduntreated mockcontrols did not contain any identifiableproteins (datanotshown).InFig.1D the direct polyacrylamide gel electrophoretic analysis ofalltheculturesconfirms thisresult in thatat2.7 mM canavanine in thepresence of0 or1% of the normalarginine concentration none of the usual proteins could be identified. A number of faint bandsnot present inthemock- infected controlswereidentified; however, they cannot beconvincingly identified inthe photo- graphs of the autoradiogram due to the close proximity of the host protein and their failureto reactwith the various humanseratested in the immunoprecipitation reaction. Examples of three suchproteins areproteins with molecular weights around 120, 102, and 49. The differential responseofvarious proteinscanagain beseenin Fig. 1D, confirming the data presented in Fig.
1A.
Proteins 138, 80, and 45 are of particular interest becausetheyarerelatively insensitiveto canavaninetreatmentandbecausecells contain- ing these proteins arepositive forEA. We(la) and others (2, 5, 6) have shown that proteins withthe sameorsimilar molecular weightsare specifically precipitated from extracts of EA- positive cells by EA+ VCA+ sera but not by EA- VCA+ sera, suggesting that indeed EA synthesis can be blocked by canavanine when thecorrectconditions arechosen.
The different effects of canavanine on the variousgroupsofproteins could be explainedas follows. Agroupofproteins (primary proteins;
la) are synthesized which exert a controlling function:whentheyaremade inanactiveform, thecellscanthensynthesize proteinsbelonging tothesecondarygroup,whereasin thepresence of 2.7 mMcanavanine and the absence ofargi-
nine, they are made in an inactive form and cannot initiate secondary protein synthesis. If, however, enough arginine is present in the medi- um a percentage of the primary proteins will be synthesized in anactive form andcaninitiatethe synthesis of secondary proteins (such as pro- teins 138, 80, and 45); if even more arginine or less canavanine is present, thena certain per- centage of the secondary proteinswill be made inanactive form andcaninitiate thesynthesisof the thirdgroupof proteins (e.g., 143).
LITERATURE CITED
1. Bayflas, G. J., and M. Nonoyama. 1978. Mechanisms of infection with Epstein-Barr virus. III. The synthesis of proteins in superinfected Raji cells. Virology 87:204-207.
la.Bayllss, G. J., and H. Wolf. 1981. The regulated expres- sion ofEpstein-Barr virus.III.Proteinsspecifiedby EBV during thelyticcycle. J.Gen. Virol.56:105-118.
2. Bodemer, W.W., W. C. Summers, and J. C. Niederman.
1980. Detectionofvirus-specific antigens in EB-(P3HR1) superinfected Rajicelisbyimmunoprecipitation. Virology 103:340-349.
3. Courtney, R. J., R. M. McCombs, and M. Benyesh- Melnlck. 1970. Antigens specified by herpesviruses. I.
Theeffect of arginine deprivation on antigen synthesis.
Virology 40:386-389.
4. Felghny, R. J., M P. Farrel, andJ. S. Pagano. 1980.
Polypeptide synthesis and phosphorylation in Epstein- Barr virus-infected cells. J. Virol. 34:455-463.
5. Kawanishi, M., K.Sugawara, and Y. Ito. 1981. Epstein- Barr virus-induced polypeptides: a comparative study with superinfected Raji, IUdR-treated and N-butyrate- treated P3HR1celis. Virology 109:72-81.
6. Mfiller-Lantzsch, N.,B.Georg, N. Yamamoto,and H.zur Hausen. 1980. Epstein-Barr virus-induced proteins. III.
Analysis of polypeptides from P3HR1-EBVsuperinfected NC37 cells by immunoprecipitation. Virology 102:231- 233.
7. Olshevsky, V.,andY. Becker.1970.Herpessimplexvirus structuralproteins.Virology40:948-960.
8. Wolf, H., and G. J.Bayliss. 1978. The role of host-virus interactions for thedevelopmentofherpesvirus-induced malignancies of new-worldprimates, p. 315-329. InP.
Chandra (ed.), Antiviral mechanisms in the control of neoplasia.PlenumPress, New York.
9. Yamamoto, N., N.Mikller-Lantzsch, and H. zur Hausen.
1980.Differential inhibition ofEpstein-Barrvirus induc- tionby the amino acid analogue 2-canavanine. Int. J.
Cancer 25:439-443.
VOL. 41,1982
