Plaque Assay of Avian Sarcoma Viruses Using Casein

Transcription

1 JOURNAL OF VIROLOGY, Sept. 1975, p Copyright Americn Society for Microbiology Vol. 16, No. 3 Printed in U.S.A. Plque Assy of Avin Srcom Viruses Using Csein PIERO C. BALDUZZI*l AND HELEN MURPHY Imperil Cncer Reserch Fund Lbortories, London, Englnd Received for publiction 17 Mrch 1975 The cseinolytic ctivity of severl strins of Rous srcom virus (RSV), conditionl nd nonconditionl mutnts of RSV, nd nontrnsforming vin leukosis viruses ws investigted. Only those viruses cpble of trnsforming chick fibroblsts in vitro induced lysis of csein incorported into n gr overly. Lysis produced distinct cler res in the turbid csein-gr gel which llowed quntittive "plque" ssy of cell trnsformtion. Csein plque formtion could not be seprted from morphologicl conversion in cultures infected by wild-type RSV strins. In pltes infected by mutnts temperture sensitive for trnsformtion, the cseinolytic ctivity ppered to be ffected by temperture to lesser extent thn morphologicl conversion. Cells trnsformed in vitro by either DNA or RNA tumor viruses produce fctor which, intercting with plsminogen contined in serum, cuses the lysis of fibrin (6). Recently Goldberg (2) found tht chicken embryo fibroblsts trnsformed by Rous srcom virus (RSV) cuse lysis of csein dded to nutrient gr medium. Wheres the ssy for fibrinolytic ctivity lends itself to ccurte quntittive studies of the verge production of plsminogen ctivtor, the cseinolytic ssy seems potentilly useful s plque ssy for vin srcom viruses. This test could lso be useful to select nonconditionl or conditionl mutnts, unble to induce proteolysis, for genetic studies with vin RNA tumor viruses. To investigte these points study ws crried out on the cseinolytic ctivity of chicken embryo fibroblsts infected with severl lbortory strins of RSV, with severl temperture-sensitive (ts) mutnts derived from these strins, nd with nontrnsforming viruses of the vin leukosis group. We hve found tht only viruses which trnsformed cells in vitro were ble to induce lysis of csein. In cells infected by mutnts of RSV, temperture sensitive for cell trnsformtion, cseinolysis ws inhibited by temperture to lesser extent thn ws morphologicl conversion. Also, in preliminry ttempts to isolte virus mutnts which would induce either morphologicl conversion or cseinolysis, these two chrcters were lwys found to be ssocited. MATERIALS AND METHODS Virus strins. Severl RSV lbortory strins of different subgroup specificity (7) were used in this I Present ddress: Deprtment of Microbiology, University of Rochester School of Medicine nd Dentistry, Rochester, N.Y investigtion: the Bryn high-titer strin of subgroup A, BH-RSV (RAV-1), of the morphr nd morph' type of morphologicl conversion (5); the Schmidt Ruppin (SR) strin lso of subgroup A; nd two Prgue strins of subgroups A nd B (PRA, PRB). Four ts mutnts isolted from PRA (8) (PRA #23, 24, 25, nd 29), kindly provided by J. Wyke, nd eight ts mutnts isolted from BH-RSV (RAV-1), three of which derived from mutgenized stocks of the morph' vrint, were lso used. The chrcteristics of these nd other mutnts isolted from BH-RSV stocks will be presented seprtely. The nontrnsforming Crr Zilber ssocited virus of ntigenic subgroup D nd trnsformtion-defective (td) derivtive isolted from the PRB strin nd kindly provided by A. Bernstein were included in these studies. Cells nd cell cultures. Secondry cultures of chick fibroblsts from C/E brown Leghorn embryos were used throughout these experiments. Cell mintennce nd conventionl infectivity ssys were crried out s previously described (1). Cseinolysis ssys. Sixty-millimeter pltes with 6 x 10" cells were infected with pproprite dilutions of different virus strins. After 45 min of dsorption the cultures were overlid with thin lyer (2 ml) of gr medium consisting of mixture of F12 medium (4) nd Dulbecco modified Egle medium in the rtio of 2:1, with 10% tryptose phosphte broth, 5% clf serum, 1.25% chicken serum, 1% dimethyl sulfoxide, 0.7% gr (Difco), nd contining s source of csein, 2% (wt/vol) dried skimmed milk (Mrvel-Cdbury Schweppes Food, Englnd). After this medium hd solidified, 5 ml of gr medium without csein were dded to ech plte. For soft gr ssys 60-mm pltes with 10" cells were infected with 1,000 focusforming units of the pproprite virus strin. After overnight incubtion the pltes were trypsinized with 1-ml volumes of trypsin (0.05%) in Tris, nd 0.2-ml volumes of cell suspension contining pproximtely 2 x 101 cells nd 100 infected cells were mixed with 2 ml of soft gr (gr 0.36%) contining csein nd distributed on pltes previously prepred with 4 ml of norml nutrient gr medium. These pltes were 707

2 708 BALDUZZI AND MURPHY usully fed with soft gr medium without csein fter 1 week. Cytotoxic plque ssy. Medi nd conditions for the plque ssy of trnsformtion defective viruses were s described by Grf (3). RESULTS Cseinolysis s n infectivity ssy. In order to investigte the potentil vlue of the cseinolytic ssy s n infectivity test for RSV, secondry cultures of chick fibroblsts were infected with seril dilutions of morphr or morph' vrints of the Bryn strin of RSV (BH-RSV) of ntigenic subgroup A, with PR nd with SR virus lso of subgroup A. After dsorption, hlf of the pltes were overlid with csein gr medium s described in Mterils nd Methods. Control cultures for conventionl focus-forming ssys were overlid with 7 ml of gr medium without csein. Pltes were incubted either t 36 C or t 41 C. Within 3 to 5 dys, distinct plques of cseinolysis ppered in pltes infected with PR nd SR virus, nd in pltes infected with the morphr vrint of BH-RSV, but not in pltes infected with the morph' vrint of the sme virus strin. Cells in pltes with csein, s well s those in pltes without csein, showed no sign of morphologicl conversion t this time. Within the next few dys, however, foci of morphologiclly trnsformed cells ppered in pltes with or without csein. Tble 1 reports the number of foci nd plques observed in n experiment 8 dys fter infection. In this, s in other experiments, the number of foci in pltes without csein ws 10 to 15% higher thn in pltes with csein. Except for morph'-infected cultures there ws high degree of coincidence of TABLE 1. Strin Cseinolytic ssy of wild-type RSV strins No csein 36C Csein No csein 41C Csein" Foci Foci Plques Foci Foci Plques BH morphr BH morph' c 77d C PRA SRA NDe ND ND Ech result is the verge number of foci or plques in duplicte 60-mm pltes infected with 0.2 ml of 10-3 dilution of ech virus. " At 36 C ll plques coincided with foci. At 41 C few plques without foci were present. c Very smll fint plques. dat 41 C morph' foci re poorly discernible. e ND, Not done. J. VIROL. plques nd foci (Fig. 1). Rre exceptions re noted in the footnote of Tble 1. On the other hnd, in pltes infected with morph' virus nd incubted t 36 C, only bout 70% of the foci produced fint cseinolytic plques, which were much smller thn those produced by foci of cells trnsformed by the other strins. At 41 C, there ws reduction of foci, ll of which developed fint cseinolytic plques. This in contrst to the other viruses where lytic plques were lrger t 41 C thn t 36 C, suggesting tht some fctor involved in the production of plques is enhnced by the higher temperture. These experiments lso show tht RAV-1, the nontrnsforming "helper" virus present in BH-RSV stocks, does not cuse proteolysis. Otherwise lrger excess of cseinolytic plques would hve been observed in pltes infected with this virus. On the other hnd, in pltes infected with BH-RSV or PR virus, occsionl well-developed foci without cseinolytic plques were observed. On the ssumption tht this chrcter could be due to virus muttions, these "non-cseinolytic" foci were isolted. The cells from individul foci were suspended in smll volume, frozen nd thwed once nd tested for bility to induce cseinolytic plques nd/or morphologicl conversion in new cultures. Out of three foci isolted from BH-RSV, two produced wildtype virus ble to cuse cseinolysis nd morphologicl conversion. One ws negtive for focus formtion nd cseinolytic plques. As foci of cells producing noninfectious virus re occsionlly found in pltes infected with high dilutions of this virus strin, it is possible tht the noninfectious suspension ws derived from one such focus. Out of two foci isolted from PR virus-infected pltes, one ws positive for both chrcters nd one showed prtil morphologicl conversion without cseinolysis. In further purifiction ttempts, however, this isolte lso produced foci of trnsformed cells with cseinolytic plques similr to those observed with wild-type virus. The resons for this behvior re not known. Possibly cellulr fctors could hve been responsible for the prtil expression of trnsformtion in the erlier tests. Control of cseinolysis. In n ttempt to investigte the role of virl versus cellulr fctors in the expression of proteolytic ctivity, experiments were crried out with four temperture-sensitive (ts) mutnts of PR virus subgroup A (PRA #23, 24, 25, nd 29) nd with eight ts mutnts isolted from BH-RSV, three of them derived from mutgenized stocks of the morph' vrint. All ts mutnts were tested t 36 C nd 41 C.

3 VOL. 16, 1975 PLAQUE ASSAY OF AVIAN SARCOMA VIRUS 709 A B FIG. 1. Pltes infected with BH RSV (RA V-i) morphr nd overlid: A, with gr nutrient medium; B, with csein-contining gr nutrient medium. Plte A received neutrl red gr on dy 8 postinfection. Pictures were tken the following dy. At 36 C the number of foci cused by the PR ts mutnts in pltes with or without csein ws roughly equl, but severl cseinolytic plques without foci could be observed t this temperture (Tble 2). The frequency of these plques vried between 10 to 30% of ll plques in different experiments. Cells from severl such res of cseinolysis without trnsformtion were isolted in smll volumes of medium, frozen, thwed, nd tested in new cultures. All suspensions tested produced 10 to 30% excess of cseinolytic res over foci of trnsformed cells s the originl ts mutnts. This suggests tht t 36 C number of infectious prticles fil to cuse morphologicl conversion while still inducing cseinolysis by the infected cells. At the nonpermissive temperture both focus formtion nd plque formtion were inhibited by more thn 90% (note the difference of inocul t the two tempertures). However, in three out of four cses production of cseinolytic res ws reduced less thn ws focus formtion. For mutnt #24, the number of residul cseinolytic plques ws pproximtely equl to the number of foci. These observtions suggest tht different.ts mutnts hve different temperture sensitivity for ech of the two prmeters of trnsformtion considered here, i.e., proteolytic ctivity nd morphologicl conversion, nd tht the former property is often more temperture resistnt thn focus formtion. With the ts mutnts of BH-RSV t 36 C ll morphr mutnts produced both foci nd cseinolytic plques with slight excess of plques. The three morph' mutnts produced welldeveloped foci of typiclly trnsformed cells with miniml or no cseinolysis. At 41 C both foci nd cseinolytic plques were strongly suppressed in ll ts mutnts with the exception of ts 38, morph' mutnt which produced very fint cseinolytic res nd no visible foci. The BH-RSV mutnts were lso tested in soft gr. For this ssy some pltes were prepred with csein medium t the time the cells were suspended in soft gr. These pltes were incubted either t 36 C or t 41 C. To other pltes csein medium ws dded 24 h fter shift in temperture from 36 to 41 C. The temperture shift ws mde fter visible colonies hd lredy ppered. In soft gr pltes t 36 C the five morphr mutnts produced compct, sphericl colonies surrounded by cseinolytic res. The morphf mutnts produced dispersed, fluffy colonies of which bout one-third showed fint cseinolytic res. In pltes incubted t the nonpermissive temperture both colony formtion nd cseinolysis were strongly inhibited. Finlly, s it would be expected from the reported temperture lbility of the plsminogen ctivtor (6), little or no cseinolysis ws observed in the temperture shift experiment. Tken ll together these experiments indicte tht the continuous expression of virus genes is required for both growth in soft gr nd cseinolysis. As the cseinolytic ctivity of ts mutntinfected, nontrnsformed cells seems to repre-

4 710 BALDUZZI AND MURPHY J. VIROL. TABLE 2. Cseinolytic ssy of PRA ts mutnts 36 C 41 C No Csein" No Strin csein csein Csein Foci Foci Plques plquesc Foci Foci Plques Foci/ PRLA #23 A PRLA #24 A PRLA #25 A PRLA #29 A Pltes incubted t 36 C were infected s in Tble 1. Pltes incubted t 41 C received 10-fold more virus. Ech result is the verge of duplicte pltes. 'In pltes with csein ech focus coincided with plque. c Clculted from pltes with csein in ech cse. sent prtil expression of trnsformtion with lower temperture sensitivity thn morphologicl conversion, the possibility ws considered tht these two prmeters of trnsformtion could be seprtely controlled by the trnsforming informtion of vin srcom viruses. In n ttempt to clrify this point, experiments were crried out to investigte whether td derivtives of vin srcom viruses would retin the bility to induce cseinolysis. Sets of pltes were infected with pproprite dilutions of stock of PRB virus, known to contin lrge mount of td virus or with td virus isolted from this stock, or with stock of Crr Zilber ssocited virus leukosis virus of subgroup D. After dsorption, duplicte pltes of ech set were overlid with csein-contining gr medium or with medium which revels res of cell lysis upon infection with td of subgroups B nd D. Pltes contining csein medium were observed t 7 nd 13 dys nd the number of cseinolytic plques with foci of trnsformed cells, plques without foci, nd foci without plques were recorded. Ares of cell lysis in the cytotoxic plque ssy nd foci ppering on these pltes were counted t dy 13. The results of such n experiment (Tble 3) show tht for PRB virus cseinolytic plques generlly pper before foci, but by dy 13 ll foci of trnsformed cells re surrounded by res of cseinolysis. Prllel pltes overlid with medium used in the cytotoxic ssy showed tht the number of cytolytic plques in this experiment ws bout 10-fold higher thn the number of foci. The pltes infected with severl dilutions of the td derivtive isolted from PRB nd those infected with Crr Zilber ssocited virus showed no cseinolytic plques while the titer of cytotoxic plques ws 6 x 107 nd 3 x 107 for the td from TABLE 3. Cseinolytic nd cytotoxic ssy of PRB RSV Cseinolytic Cytotoxic ssy ssy Dy - Plques Plques Foci Plques Foci + Foci NDb ND Pltes were infected with pproprite dilutions of PRB RSV nd overlid with csein medium or with medium for the cytotoxic ssy. The results re the sum of counts on triplicte pltes. "ND, Not done. PRB nd Crr Zilber ssocited virus, respectively. DISCUSSION These results show very high correltion between morphologicl conversion nd the proteolytic ctivity of virus-infected cells. Neither viruses which nturlly do not trnsform cells in vitro (leukosis viruses) nor td derivtives of srcom viruses (ts mutnts of the PR strin or of the repliction-defective BH-RSV strin, nd td vrints of PRB) induce cseinolytic ctivity. This correltion is unffected by the group specificity (A, B, or D) of the virus. The cseinolytic plque ssy could be used s n infectivity ssy for RSV nd possibly for other vin viruses ble to trnsform cells in vitro, such s the vin myeloblstosis virus (C. Moscovici, L. Gzzolo, nd M. G. Moscovici, personl communiction), lthough it offers little or no dvntge over the conventionl focus ssy. In the cse of ts mutnts, however, the cseinolytic ssy reveled infectious virus unble to cuse cler morphologicl con-

5 VOL. 16, 1975 version. It seems tht the ssy could be useful for gents tht nturlly do not produce esily detectble foci. Both in the cse of BH-RSV morph' nd in three of the four ts mutnts of PRA RSV morphologicl conversion seems to be more temperture lbile thn cseinolysis. In the other ts mutnt, however, both ctivities were pproximtely eqully reduced t 41 C. Even in this cse both ctivities were mrkedly reduced by the muttion. Occsionlly, t 36 C, foci were found which were not ccompnied by cseinolytic plques, but when virus ws isolted from such trnsformed foci it hd the property of inducing cseinolysis. Tken ll together, the simplest interprettion of these results is tht single virus gene is responsible for both morphologicl conversion nd cseinolytic ctivity s well s for the bility to grow in soft gr. The expression of these chrcters, however, is ltered to somewht different extent by temperture, nd lso perhps by undefined host cell functions. ACKNOWLEDGMENTS We wish to thnk A. R. Goldberg for mking vilble technicl detils of the cseinolytic ssy prior to publiction. P. C. Blduzzi is grteful to M. Stoker, R. A. Weiss, nd G. S. PLAQUE ASSAY OF AVIAN SARCOMA VIRUS 711 Mrtin for their hospitlity t the Imperil Cncer Reserch Fund Lbortories. This work ws supported by Ntionl Cncer Institute Fellowship CA nd by Public Helth Service reserch grnt CA from the Ntionl Cncer Institute. LITERATURE CITED 1. Blduzzi, P., nd H. R. Morgn Mechnisms of oncogenic trnsformtion by Rous srcom virus. Intrcellulr inctivtion of cell trnsforming bility of Rous srcom by 5-bromodeoxyuridine nd light. J. Virol. 5: Goldberg, A. R Incresed protese levels in trnsformed cells: csein overly ssy for the detection of plsminogen ctivtor production. Cell 2: Grf, T A plque ssy for vin RNA tumor viruses. Virology 50: Hm, R. G Clonl growth of mmmlin cells in chemicll defined synthetic medium. Proc. Ntl. Acd. Sci. U.S.A. 53: Temin, H. M The control of cellulr morphology in embryonic cells infected with Rous srcom virus in vitro. Virology 10: Unkeless, J. C., A. Tobi, L. Osswiski, J. D. Quigley, D. B. Rifkin, nd E. Reich An enzymtic function ssocited with trnsformtion of fibroblsts by oncogenic viruses. J. Exp. Med. 137: Vogt, P. K Envelope clssifiction of vin RNA tumor viruses, p In R. M. Dutcher (ed.), Comprtive leukemi reserch, Krger, Bsel. 8. Wyke, J. A., nd M. Linil Temperture-sensitive vin srcom viruses: A physiologicl comprison of twenty mutnts. Virology 53: Downloded from on Jnury 7, 2019 by guest