Middle T Antigen as Primary Inducer of Full Expression of

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1 JOURNAL OF VIROLOGY, JulY 1980, p X/80/07-019/ 14$0.00/0 Vol. 35, No. I Middle T Antigen s Primry Inducer of Full Expression of the Phenotype of Trnsformtion y Polyom Virus YOSHIAKI ITO,t* NIGEL SPURR, AND BEVERLY E. GRIFFIN Imperil Cncer Reserch Fund, Lincoln's Inn Fields, London WCA 3PX, Englnd A lrge numer of polyom virus-trnsformed cells of rt, mouse, nd hmster origin were exmined for presence of T-ntigen species. The results showed tht ll lines of cells contined middle nd smll T ntigens, ut not ll contined full-sized lrge T ntigen; in some cell lines lrge T ntigen ws sent, wheres in others it ws present s truncted forms lcking vrious lengths of the croxyterminl prt of the protein. Cells trnsformed y the new vile deletion mutnts of polyom virus, dl-8 nd dl-3, formed lrger nd smller colonies or foci, respectively, when they were suspended in semisolid medium or plted s monolyers together with untrnsformed cells on plstic surfce. The deletions in the DNA of these mutnts resulted in the shortening of the lrge nd middle T ntigens simultneously without ffecting the size of the smll T ntigen. Vrition of lrge T-relted proteins in dl-8- nd dl-3-trnsformed cells seemed to e the sme s tht oserved in wild-type-trnsformed cells. Regrdless of the mount nd size of lrge T-relted protein in mutnt-trnsformed cells, the phenotype of the cells ws entirely dependent on the mutnt used. The results suggest tht (i) persistence of lrge T ntigen is not universlly required for the mintennce of the trnsformtion phenotype, (ii) smll T ntigen lone my not e sufficient for inducing the full expression of the trnsformtion phenotype, nd (iii) middle T ntigen is implicted s eing primrily responsile for the full expression of the phenotype of trnsformtion. The results lso provide the evidence tht the croxy-terminl region of middle T ntigen nd prt of lrge T ntigen re encoded in the genome in the sme DNA segment round mp units 88 to 94 in different reding frmes. Recent studies on trnsforming gene products of ppovviruses hve mde it possile to ddress the question, in moleculr terms, of the function of individul products in ltering cells to trnsformed stte. At lest two proteins of pprent moleculr weights of 81,000 to 108,000 nd 17,000 to,000 (designted lrge nd smll T ntigens, respectively) re generlly encoded in trnsforming genes of ppovviruses (4, 19, 9). In the cse of polyom virus, third trnsforming gene product, designted middle T ntigen, hs een identified (14, 17, 30, 31). This protein ws first found in ssocition with the plsm memrne of productively infected mouse cells (18). The oservtion tht nontrnsforming mutnt of polyom virus, NG-18 ( prototype of hr-t mutnts []), ws unle to induce middle T ntigen (17, 18) stimulted us to study the possile iologicl roles of this protein nd the significnce of its memrne ssocition in cell trnsformtion. tpresent ddress to which reprint requests should e sent: Recominnt DNA Reserch Unit, Ntionl Institute of Allergy nd Infectious Diseses, Ntionl Institutes of Helth, Bldg. 5, Rm. 39, Bethesd, MD 005. Studies on hr-t mutnts hve shown tht they lck the ility to direct the synthesis of functionl smll T s well s middle T ntigens (13, 15, 17, 19, 6, 34), lthough they re le to induce norml lrge T ntigen (0, 6). The hrt mutnts, unlike wild-type virus, hve lso een shown to induce no morphologicl chnge in cutely infected rt cells (7). A line of cells hs een isolted which contins integrted s well s free hr-t genomes (4). These cells re indistinguishle from untrnsformed prentl cells y morphologicl nd other criteri. Only lrge T ntigen is detected in these cells. Since the presence of free virl DNA in trnsformed cells hs een shown to e dependent on the tsa gene function (35) (which, together with the hr-t gene function, constitutes the two known mutully complementry erly gene functions [6, 7]), the lrge T ntigen, product of the tsa gene, is presumed to e functionl in these cells. In spite of the fct tht the trnsformed stte of some lines of trnsformed rt 3T3 cells hs een reported to e tsa gene dependent (, 8), the results descried ove suggest tht, generlly, lrge T ntigen lone is not sufficient for induc- 19

2 0 ITO, SPURR, AND GRIFFIN ing the trnsformtion phenotype nd tht the middle or smll T ntigens or oth my e more importnt for its expression. It hs lso een reported tht polyom virus DNA which is cleved in the middle of the tsa gene not only retins ut lso cquires n incresed ility to induce tumors in hmsters (16). Although it is not cler why enhncement of tumorigenicity of the DNA occurs y the interruption of the tsa gene, this oservtion is consistent with the results descried ove. In the studies descried in this pper, we provide the evidence which suggests tht middle T ntigen, rther thn smll T ntigen, ppers to e essentil for inducing the full expression of the trnsformtion phenotype. MATERIALS AND METHODS Viruses. Wild-type polyom virus A nd A3 strins (10), the temperture-sensitive mutnt of polyom virus, ts- (9), nd newly isolted vile deletion mutnts, dl-8 nd dl-3 (1), were plque purified nd grown t 3 C in primry y mouse kidney cells in Dulecco-modified Egle minimum essentil medium (E4) supplemented with 10% horse serum. Cells nd culture medium. Mouse 3T6 cells were used s permissive cells for productive infection y the virus. Cells were grown in E4 supplemented with 5% fetl clf serum. After cells were infected with the virus, they were kept in E4 contining 3% horse serum. Prentl strins of cells which were trnsformed y the viruses re primry whole rt emryo (WRE) firolsts from Wistr/Furth strin; n estlished line of rt firolst from Fischer strin, F408 (8); rt 3T3 line 3Y1 from Fischer strin (3); estlished mouse cell lines BALB/c 3T3(A31) nd Swiss 3T3; nd n estlished line from hmster, NIL 8 (5). Polyom virus-trnsformed Swiss 3T3 cells, Py6, were otined from T. L. Benjmin (Hrvrd Medicl School, Boston, Mss.). BALB/c 3T3(A31) cells trnsformed y ts-c ( lte mutnt), ts-c-1, were otined from M. Fried (Imperil Cncer Reserch Fund, London, Englnd). Cells were cultured in E4 supplemented with 5% fetl clf serum. Trnsformtion. BALB/c 3T3(A31) cells were infected with wild-type, dl-8, or dl-3 viruses t multiplicity of out 10 nd 100 PFU per cell. After dsorption for 90 min, undsored virus ws removed, nd E4 contining 5% fetl clf serum ws dded. After the infected cells hd een incuted for out 18 h t 37 C, the medium ws chnged to E4 contining 5% fetl clf serum nd 400 hemgglutintion inhiition units of neutrlizing ntiserum ginst polyom virus per ml (otined from L. V. Crwford). The cells were kept under these conditions t 37 C with weekly medium chnge, using the sme medium. Dense foci of trnsformed cells ppered in wild-type virus nd dl- 8-infected cultures in weeks. In dl-3-infected cultures, smll, slowly growing foci ecme visile fter further 1 to weeks of incution. Severl foci were picked (one clone per dish), recloned, nd kept in culture in the presence of neutrlizing ntiodies. F408 rt cells were infected with wild-type, dl-8, or J. VIROL. dl-3 viruses t multiplicity of out 10 PFU/cell. After dsorption (90 min t 37 C), infected cells were incuted in E4 contining 5% fetl clf serum for 4 h. Cells were then trypsinized nd replted t cell density of out 105 cells per 50-mm dish nd incuted with E4 contining 5% fetl clf serum with weekly medium chnge or suspended in soft gr (5 x 104 cells per 50-mm petri dish) (5). As in the cse of mouse cells, dense foci ppered in out weeks in wild type- nd dl-8-infected cultures. For dl-3- infected cells, smll foci ecme visile only fter 1 to weeks of further incution. Severl clones were isolted, recloned, nd kept in culture. Trnsformtion of other cells were performed similrly. Extrction nd seprtion of [35S]methionineleled T ntigens. The procedures to extrct nd seprte T ntigens from productively infected 3T6 cells hve een descried previously (17). Trnsformed cells in 50-mm dishes (c. x 106) were leled for 4 h in ml of E4 lcking methionine nd contining 300,uCi of [35S]methionine. A 30-,ld mount of nti-t serum ws usully sufficient to isolte the mximum mount of T ntigens from the extrct of cells from 50-mm petri dishes. Fingerprint nlysis of T ntigens. [3"S]methi-. onine-leled T ntigens induced y dl-8, dl-3, nd wild-type viruses were isolted from infected 3T6 cells (107) leled etween 7 to 31 h fter infection with mci of [3S]methionine nd seprted y electrophoresis in polycrylmide gels contining sodium dodecyl sulfte. In the cse of trnsformed cell T ntigens, x 107 cells were leled with 4 mci of [35S]methionine. The gels were dried immeditely fter run without fixtion. Positions of T-ntigen species were visulized y exposing the dried gels to X-ry film. The portions of gels contining T ntigens were cut out. The elution of T ntigens from gels, performic cid oxidtion nd trypsin digestion of proteins, nd two-dimensionl seprtion of tryptic peptides on cellulose-coted glss pltes (0 y 0 cm) y electrophoresis t ph 4.5 for the first dimension nd chromtogrphy for the second dimension were descried elsewhere (17). Rdioctivity pplied to single plte ws 5 x 103 to x 104 cpm, nd exposure time ws 5 to 0 dys. In mixing experiments, pproximtely the sme numer of counts were comined. RESULTS T ntigens induced y the mutnts dl-8, nd dl-3. Two new vile mutnts of polyom virus, dl-8 nd dl-3 (which hve deletions in the erly region of polyom virus genome etween 88.0 to 91.5 nd 91.5 to 94.5 mp units, respectively), hve een isolted (1). In preliminry chrcteriztion of the mutnts, it ws found tht in productive infection dl-8 ws inefficient in virl DNA repliction, wheres dl-3 ws t lest s efficient s wildtype virus. The most interesting fetures of the mutnts were tht they ppered to hve ltered trnsformtion properties. Cells trnsformed y dl-8 virus often produced lrger foci in monolyers in liquid medium nd lrger colonies in

3 VOL. 35, 1980 soft gr thn those trnsformed y wild-type virus. Mutnt dl-3 induced very smll foci of trnsformed cells in monolyers of infected cells in liquid medium ut did not seem to induce trnsformed cells cple of growing in soft gr. It ws therefore interesting to sk which of the virl gene products ws responsile for these ltered phenotypes since the deletions were in different mp positions from those of ny other polyom virus erly mutnts known to ffect cell trnsformtion. T ntigens induced in productively infected cells y the deletion mutnts nd y wild-type virus re shown in Fig. 1. Mutnt dl-8 produced lrge T ntigen which ws smller thn tht of the wild-type protein. The pprent difference in moleculr weight, estimted y sodium dodecyl sulfte-polycrylmide gel electrophoresis, ws 5,000. This difference grees well with the size of the deletion in the mutnt (out %), ssuming tht the deleted DNA codes for lrge T ntigen. In ddition, the mutnt induced shortened version of middle T ntigen, 50K protein (Fig. 1) (see elow for peptide mp nlysis). The fct tht the pprent differences in size of lrge nd middle T ntigens of wild-type virus nd the dl-8 mutnt re lmost exctly the sme, nmely 5,000 dltons, strongly indicted tht the re etween 88 nd 91.5 mp units in the erly region codes for oth lrge nd middle T ntigens. The mutnt dl-3 lso induced lrge T ntigen which ws smller thn the corresponding wild-type protein. In this cse lso, the reduction of the pprent size of the lrge T ntigen (out 5,000 dltons) ws tht expected from the size of the deletion, indicting tht the region etween mp units 91.5 to 94.5 codes for lrge T ntigen. The size of the middle T ntigen, however, ws much smller thn tht expected from the size of the deletion. Mutnt dl-3 did not induce 55K protein, ut induced new protein with n pprent moleculr weight of out 43,000. The smll T ntigen induced y these two mutnts ws indistinguishle in size from tht of wildtype protein, s shown in Fig. 1. Fingerprints of methionine tryptic peptides of T ntigens induced y the mutnts dl-8 nd dl-3. Methionine-contining tryptic peptides of T ntigens of wild-type virus nd mutnts dl-8 nd dl-3 were nlyzed y twodimensionl fingerprinting. As shown in Fig. A, wild-type lrge T ntigen gives six mjor nd four minor peptides, which re consistently found in ll experiments. They re leled 1 through 10 in the figure. As pulished erlier, peptides 1 nd re shred y ll three T- ntigen species (17). The other peptides re unique to lrge T ntigen. Figures B nd C MIDDLE T ANTIGEN OF POLYOMA VIRUS 1 dl-8 lrge T- di-8 middlet- c d o -LrgeT " dl-3 lrge T - -Middle T -di-3 middle T - -Smll T FIG. 1. T ntigens induced y mutnts dl-8 nd dl-3 nd wild-type virus in productively infected cells. 3T6 cells ( x 1P) were infected with either dl- 8, dl-3, or wild-type viruses nd leled with [35S]- methionine. T ntigens were isolted nd seprted y sodium dodecyl sulfte-polycrylmide gel electrophoresis. The gel used hd 5% stcking gel (I cm in length) on top of 17-cm 10%7 gel. Electrophorests ws crried out t 100 V nd terminted when the dye mrker (romophenol lue) reched out 1 cm from the ottom. The gel ws fixed, stined, fluorogrphed, nd exposed to X-ry film for dys s descried erlier (18). () Mock-infected cells; () dl- 8-infected cells; (c) wild type-infected cells; (d) dl-3- infected cells. The nd t the 45K position which is present in ll infected cells nd is not ffected y the deletions is virus cpsid protein, VPI, nd. show tht the lrge T ntigens of mutnts dl-8 nd dl-3 seem to contin ll the peptides found in the wild-type protein even though the sizes of the mutnt proteins re pproximtely 5% smller thn tht of the wild-type species. Figure D shows the fingerprints of the mixture of lrge T ntigens of dl-8 nd wild type, nd Fig. E shows those of dl-3 nd wild type. There is no evidence to indicte ny difference in fingerprints mong these lrge T ntigens. A methionine-leled tryptic peptide fingerprint of middle T ntigen of wild-type virus is shown in Fig. 3A. Peptides designted 1 nd re found lso in lrge nd smll T ntigens, s mentioned ove. The peptides designted nd re lso present in smll T ntigen (17). The rest of the peptides re unique to middle T ntigen. The fingerprint of the protein of dl-8 with n pprent moleculr weight of 50,000 is

4 ITO, SPURR, AND GRIFFIN J. VIROL. A B C _ v* p 5 4 EB WT D 4 WT+ dl-8 di E WT- dl-3 FIG.. Two-dimensionl tryptic peptide fingerprints of lrge T ntigens. (A) Wild type; (B) dl-8; (C) dl-3; (D) wild type mixed with dl-8; (E) wild type mixed with dl-3. Spots re numered s descried erlier for the fingerprint otined from lrge T ntigen synthesized in ts--infected cells which were leled t the nonpermissive temperture fter the culture ws shifted up from thepermissive temperture t lte in infection (1 7). There re some vritions in intensity of the lel, prticulrly of the spot numered 6. This is one of the mjor spots in ts- lrge T ntigen leled t the nonpermissive temperture, wheres it is one of the minor spots in wild-type lrge T ntigen. 4w di shown in Fig. 3B. The fingerprint is similr to tht of wild-type middle T ntigen (Fig. 3A) nd clerly different from tht of lrge T ntigen (Fig. ), confirming tht the 50,000-dlton protein is relted to middle T ntigen. The peptide designted u' in the fingerprint of dl-8 middle T ntigen differs from the peptide designted u in the wild-type fingerprint. The difference is est seen in the fingerprint of the mixture of the two middle T preprtions (Fig. 3D). The peptide designted v of wild-type middle T is missing in the middle T of dl-8. Therefore, t lest two peptides chrcteristic of wild-type virus middle T re missing, nd t lest one chrcteristiclly new peptide is present in the middle T ntigen of dl-8. The fingerprint of the protein of the mutnt dl-3 with n pprent moleculr weight of 43,000 is shown in Fig. 3C. In spite of the fct tht the pprent moleculr weight of the protein is out 0% smller thn tht of wild-type virus middle T ntigen, no differences re oserved in fingerprints of the two proteins (Fig. 3E). The fingerprints of smll T ntigens of wildtype virus nd mutnts dl-8 nd dl-3 were exmined similrly nd were found to e indistinguishle (dt not shown). As will e discussed lter in conjunction with Fig. 9, these dt, together with the results in Fig. 4 nd 5 elow, re importnt in determining the coding region nd the reding frme for the croxy-terminl region of middle T ntigen. T ntigens in polyom virus-trnsformed cells. As pointed out erlier, the T ntigens present in polyom virus-trnsformed cells vry in size, numer, nd mount from cell line to cell line (17). Severl reports hve een pulished which del with limited chrcteriztion of polyom virus T ntigens in trnsformed cells or in cells derived from tumors. The dt show, mong other things, tht some lines of cells contin lrge T ntigen nd others do not (15,

5 VOL. 35, 1980 MIDDLE T ANTIGEN OF POLYOMA VIRUS 3 A B v C u ui u w go w d w v 1 lw w WT x 0 y D WT + di-8 w di-8 UI E WT + di-3 FIG. 3. Two-dimensionl tryptic peptide fingerprints of middle T ntigens. (A) Wild type; (B) dl-8; (C) dl- 3; (D) wild type mixed with dl-8; (E) wild type mixed with dl-3. Spots re mrked s descried erlier for ts middle T ntigen (17). Although there re severl minor spots, those which re most consistently oserved oth in ts- nd wild-type proteins re mrked. Two rrows in B indicte the positions of the spots u nd v which re missing in the mutnt middle T ntigen ut present in wild-type middle T ntigen (A). 16, 0). However, none of these reports hs estlished conclusively the generl structurl fetures of T ntigens in trnsformed cells. Since we noticed enormous vritions in the generl T-ntigen pttern from cell line to cell line, we decided to screen lrge numer of trnsformed cells derived from three different species of rodents with the id of n improved detection procedure which reduced the ckground sustntilly (17). To fcilitte the detection of middle nd lrge T ntigens, dl-8-trnsformed cells were lso included in the screening experiments. Tle 1 summrizes the results of such experiments. All of the 50 lines of cells tested contined proteins indistinguishle in size from the middle nd smll T ntigens induced in productively infected cells. When cells were trnsformed y the mutnt dl-8, the chrcteristiclly shortened middle T-like protein ws present. Numers in prentheses in Tle 1 re those determined with the dl-8 mutnt. Thirty-two out of 50 cell lines contined protein of the size of lrge T ntigen. Of the other 18 cell lines, 3 ; w v -w di-3 y lines (1 of rt [dl-8] nd of hmster [A3, dl-8] origin) did not hve ny component other thn middle nd smll T ntigens t the detectle level. The rest of the cell lines, which include ll of the four mouse cell lines tested, contined protein or proteins rnging in size from 8K to over 150K, which did not comigrte with ny of the three T-ntigen species. Three out of the 3 lines which contined protein of the size of lrge T ntigen lso hd other components rnging from 36K to 39K. Selected exmples of these vrious protein species were nlyzed y peptide mpping. The results of the nlyses led us to conclude tht the protein ptterns shown in Fig. 4 re typicl exmples of the T ntigen species present in polyom virus-trnsformed cells. The cells, trnsformed mouse cells, contined three mjor species of proteins. A look protein ws not induced in Py6 cells (Fig. 4A) ut, insted, protein with n pprent moleculr weight of 70,000 ws found s shown. The two other proteins in Py6 cells were very similr in electrophoretic moility to the middle nd smll T l0 y

6 ..W 4 ITO, SPURR, AND GRIFFIN J. VIROL. TABLE 1. Lrge, middle, nd smll T ntigens ofpolyom virus present in vrious cell lines trnsformed y wild-type or mutnt viruses' T ntigen Prentl cells Isolted from Numer tested L M S F408 Soft gr 1 (13) 17 (11) 1(13) 1(13) F408 Dense foci 8 (4) 6 (3) 8 (4) 8 (4) 3Y1 Dense foci WRE Dense foci WRE Tumor 1 NIL8 Softgr 9 (6) 6 (5) 9 (6) 9 (6) BALB/c 3T3 Dense foci Swiss 3T3 Soft gr T ntigens of ech line of cells were nlyzed y sodium dodecyl sulfte-polycrylmide gel electrophoresis s descried in the text. Numers under L (lrge T ntigen), M (middle T ntigen), nd S (smll T ntigen) re the numer of cells which contined the protein with the sme electrophoretic moility s tht of lrge, middle, or smll T ntigens isolted from productively infected cells. Of these numers, those otined from dl-8- trnsformed cells re shown in prentheses. Two lines originting from WRE cells isolted s dense foci (ts/ RE/B nd ts/re/b3) re trnsformed y the mutnt ts (9). The cell lines do not show temperturedependent ltertions of trnsformtion phenotype, nd neither of them contins full-sized lrge T ntigen. Mjor species of T ntigen in these cells re 43K nd 37K proteins, respectively. One of the BALB/c 3T3 lines is trnsformed y the mutnt ts-c (M. Fried, unpulished) nd provided y M. Fried. This line does not contin full-sized lrge T ntigen ut, insted, contins protein of over 150K. Prentl cells re descried in the text. A B C D F m I wp=.s- Lrge T K Lrge T- Middle T- - A -55 K -80 K 0*0 - Mm -60 K Middle T- -55 o- -50 K - - Smll T- _ -K T T C Smll T- - K T T C T T C FIG. 4. Polyom virus T ntigens in trnsformed mouse cells. [3'SJmethionine-leled T ntigens of polyom virus-trnsformed mouse cells were isolted nd seprted in 1Oi. polycrylmide gels. (A) T ntigens from productively infected 3T6 cells; (B) Py6 cells; (C) Py/A31/C-1 cells; (D) dl-8/a31/xxa cells. Positions of lrge, middle, nd smll T ntigens of the wild type from productively infected cells in lnes prllel to those of (C) nd (D) re indicted. T, Anti- T serum treted; C, Control serum treted. ntigens found in productively infected mouse cells. Py/A31/C-1 (Fig. 4B) nd dl-8/a31/ XXA cells (Fig. 4C) lso did not hve look proteins ut contined 80K nd 60K proteins, respectively. The former contined proteins tht comigrted with middle nd smll T ntigens. The ltter contined protein tht comigrted with smll T ntigen nd the 50K protein which comigrted with the chrcteristic dl-8 middle T ntigen. The fingerprint of the 70K protein of Py6 cells (Fig. 5, IB) clerly shows this protein to e relted to lrge T ntigen (Fig. 5, IA), ut three mjor peptides unique to lrge T ntigen re missing in the fingerprint of this protein. As

7 VOL. 35, 1980 I II MIDDLE T ANTIGEN OF POLYOMA VIRUS 5 III T t 0 o A B C 1 v w_ _ 6 A B C w xo v v w_ e X y Xe-#y, y 1 l A I 0 lid FIG. 5. Two-dimensionl trypticpeptide fingerprints of[35s]methionine-leled 70K, 55K, nd Kproteins of Py6 cells nd those of lrge (I), middle (II), nd smll (III) T ntigens of wild-type virus induced in productively infected 3T6 cells. (IA) Lrge T ntigen ofproductively infected cells; (IB) 70K protein of Py6 cells; (IC) mixture ofia nd IB; (IIA) middle T ntigen ofproductively infected cells; (IIB) 55Kprotein of Py6 cells; (IIC) mixture ofiia nd IIB; (IIIA) smll T ntigen ofproductively infected cells; (IIIB) K protein of Py6 cells; (IIIC) mixture of IIIA nd IIIB. B C descried erlier, the peptides numered 1 nd re shred y ll three T ntigen species nd re considered to e derived from somewhere etween 74 nd 80 mp units on the polyom virus DNA (17). The fct tht these two peptides re lso present in the 70K protein suggests tht the sequence (equivlent to c. 30,000 dltons) of lrge T ntigen which is missing in the 70K protein is lrgely (nd possily entirely) locted t the croxy-terminl end of the protein. This implies tht peptides designted 4, 7, nd 9 proly come from the region ner the croxyterminus of the protein. Figure 5, IIB, shows the fingerprint of the tryptic peptides derived from the 55K protein of Py6. This protein is very similr to the middle T ntigen present in productively infected cells (Fig. 5, IIA). The mixing experiment of the wildtype nd Py6 55K peptides shows, however, tht there is smll difference (Fig. 5, IIC). The peptide of the middle T ntigen from productively infected cells designted (y) is not present in the 55K protein of Py6 nd new peptide designted (y') is seen. At present, the reson for C S

8 6 ITO, SPURR, AND GRIFFIN this smll difference is not cler ut my reflect virus strin differences, since the strin of the virus used for Py6 cells y T. Benjmin is likely to e different from the A3 strin of wild-type virus used here. Figure 5, III, shows the fingerprints of the tryptic peptides derived from the K protein of Py6 cells nd tht isolted from productively infected cells. There is no notle difference etween them. Severl proteins in other trnsformed cells which comigrted with middle nd smll T ntigens were lso nlyzed y peptide mpping. The fingerprints were indistinguishle from those of the middle nd smll T ntigens produced in productively infected cells. The peptide (y') in Fig. 5, IIB, does not pper to e unique to trnsformed cells (dt not shown). The 50K protein of dl-8/a31/xxa cells (Fig. 4C) ws confirmed y peptide nlysis to e the chrcteristiclly shortened dl-8 middle T ntigen (dt not shown). A C -i 3 1 J. VIROL. Severl further exmples of the peptide mps of the truncted forms of lrge T ntigens with different sizes re shown in Fig. 6. Two of them re derived from the 80K nd 60K proteins shown in Fig. 4B nd C. The 43K nd 37K proteins were isolted from trnsformed rt emryo cells. Like the 70K protein of Py6 cells, ll of them contined the peptides designted 1,, nd 3. A further point to note is tht the smller the protein, the smller the numer of peptides unique to the lrge T ntigen present in the fingerprint. The significnce of this will e discussed lter. Phenotype nd T ntigens of cells trnsformed y the mutnts dl-8 nd dl-3. To chrcterize further the trnsformtion properties of dl-8 nd dl-3 nd to exmine T ntigens present in cells trnsformed y these mutnts, mouse nd rt cells were trnsformed y the mutnts s well s y wild-type virus. A numer of clones of trnsformed cells were isolted. B D i 1-3 FIG. 6. Methionine-contining tryptic peptide fingerprints of 80K, 60K, 43K, nd 39K proteins from four different lines of trnsformed cells. (A) 80K protein from Py/A31/C-1 cells (Fig. IB); (B) 60K protein from dl- 8/A31/XXA cells (Fig. 1C); (C) 43K protein from ts/wre/b cells (Tle 1); (D) 37K protein from ts/ WRE/B3 cells (Tle 1). The spot 3 in D hs migrted slightly normlly. However, this peptide ws indistinguishle from uthentic peptide 3 in mixing experiment (dt not shown).

9 VOL. 35, 1980 From mouse cells infected with dl-8 nd dl-3, lrger, denser foci or much smller nd more slowly growing foci, respectively, were otined thn were seen with cells infected y wild-type virus (dt not shown) s ws the cse with rt cells (1). To determine whether the reduced ility of dl-3 to induce stndrd-sized foci is due to n inefficient expression of some erly functions tht precede gene expression of the mutnt or whether it is due to impired gene(s) tht re expressed in trnsformed cells, severl clones of mutnt dl-3-trnsformed rt nd mouse cells were grown nd replted onto culture dishes fter eing mixed with n excess of untrnsformed norml cells. Upon replting, dl- 3-trnsformed cells were still found to produce much smller, more slowly growing foci thn wild-type trnsformed cells (Fig. 7 nd 8). These trnsformed cells were lso replted in soft gr. Tle shows the plting efficiencies of the cells from ech of severl lines of trnsformed rt cells in soft gr. All of three clones ech of the dl-8- nd wild type-trnsformed cells tested grew well in soft gr nd formed visile colonies in 9 dys of incution t 37 C. Plting efficiencies vried from to 77% (Tle ). On the other hnd, dl-3-trnsformed cells hd not formed mcroscopiclly visile colonies in soft gr in 9 dys. By microscopic exmintion, however, it could e seen tht these cells divided severl times during the period. After further week of incution, very smll colonies of dl-3- trnsformed cells ecme mcroscopiclly visile. Plting efficiencies were clculted sed on numers of microscopiclly oservle colonies t 9 nd 17 dys fter plting, nd they re given in prentheses in Tle. Similr nlysis of nontrnsformed (control) cells showed tht less thn 0.% of the cells divided to form microscopic colonies. Although there re vritions in the plting efficiencies oserved (5 to 43%, it ws concluded tht dl-3-trnsformed cells cn divide in soft gr under the conditions used. However, the increse of the size of the colonies is quite significntly slower thn oserved with dl-8- nd wild type-trnsformed cells. The properties of these cloned cells seemed to e essentilly unltered fter eing mintined for t lest 6 months in culture. Therefore, it is likely tht the much slower growth of colonies of dl- 3-trnsformed cells, or the fster growth of dl- 8-trnsformed cells, in soft gr or of foci on plstic surfces in liquid medium in popultion of nontrnsformed cells is due to muttion in virl gene(s) which is eing expressed in these trnsformed cells. T ntigens of wild type- nd dl-8-trnsformed cells listed in Tle were nlyzed nd included in the summry results in Tle 1. All three lines MIDDLE T ANTIGEN OF POLYOMA VIRUS 7 of wild type-trnsformed cells contined lrge, middle, nd smll T ntigens of norml size. Two of the dl-8-trnsformed cell lines contined chrcteristiclly shortened lrge nd middle T ntigens (see Fig. 1), wheres one of the lines, clone Z, did not contin detectle level of lrge T or lrge T-relted proteins. T ntigens in dl-3-trnsformed cells were lso exmined. The generl T-ntigen pttern in these cells ws essentilly the sme s tht descried in Tle 1 for wild type- nd dl-8-trnsformed cells. Tht is, ll the lines contined smll T nd chrcteristic dl-3 middle T ntigens. Two of these lines contined lrge T ntigen chrcteristic of dl- 3 (clones nd 1). The other three lines contined shorter proteins pproximtely 85K (clone 3), 75K (clone 13), nd 45K (clone 14) in size (dt not shown). DISCUSSION The results descried in this pper provide the evidence on the iologicl significnce of middle T ntigen s well s definitive evidence concerning the region within the polyom virus genome tht codes for the unique prt (croxyterminl hlf) of middle T ntigen. Erlier, the three known species of T ntigens were shown, y peptide nlysis, to shre common polypeptides which were ssigned to the mino-terminl region. The croxy-terminl regions of the three ntigens were found to e unique (17, 31). The peptide mp nlyses of wild-type virus proteins, however, did not llow the croxyterminl hlf of middle T ntigen to e mpped to ny specific region of the genome. The results in Fig. 1 show tht the deletions of the DNAs of dl-8 nd dl-3 shorten lrge nd middle T ntigens simultneously. The fct tht the pprent differences in size of lrge nd middle T ntigens of wild-type virus nd the mutnt dl-8 re lmost exctly the sme, nmely 5,000 dltons, strongly indictes tht the re etween 88 nd 91.5 mp units in the erly region codes for oth lrge nd middle T ntigens. In the cse of dl-3, the reduction of the size of lrge T ntigen is tht expected from the size of the deletion. However, the reduction of the size of middle T ntigen is much greter thn expected. A possile explntion for this is tht the mino cids present in wild-type middle T ntigen nd sent in the mutnt my e cusing normlly slow migrtion in sodium dodecyl sulfte-polycrylmide gels since mrna's of the mutnt hve no nomly other thn the deletion (R. Kmen, personl communiction). Since the deletions of the DNAs of dl-8 nd dl-3 do not ffect the size of smll T ntigen, the positions of the deleted DNAs re locted

10 8 ITO, SPURR, AND GRIFFIN J. VIROL. C~. S ". Q to w3 '''.C 0_> - /- s -* X -, / /..., ' =_. I.'..,}.. 4'*.,. K1,N /---s".1 'I ii 0, ( \!. Y S -5 " e o A, ~ z L.X Cot Q ~o' ocl > 00 ; - 6-~- 4 tts~ * okt o X LAO S.- I C,) 0 0 C?N

11 VOL. 35, 1980 Im C d16 'I~ ~ f '.' d13 FIG. 8. Higher mgnifiction ofdl-3-trnsformed cell foci compred with those of dl-8. Top dishes, 7 dys; ottom dishes, 1 dys. eyond the croxy terminus of smll T ntigen, nd they seem to encode the unique region of lrge nd middle T ntigens. This suggests tht middle nd lrge T ntigens re encoded in different reding frmes in the re where the deletions lie. The mino cid sequences predicted from the two open reding frmes of DNA sequences in the region etween 86 nd 99 mp units were shown erlier (3). The sequence thought to e prt of lrge T ntigen hd only one methionine-contining tryptic peptide. The sequence predicted for the unique region of middle T (which contins row of hydrophoic mino cids ner the predicted croxy terminus flnked on either side y hydrophilic mino cids) contins 10 methionine residues which reside within 7 methionine tryptic peptides. There is no methionine-contining tryptic peptide which should e ffected y the deletion in mutnt dl-8 in lrge T ntigen, wheres there re two methionine tryptic peptides predicted to lie within middle T ntigen in the region deleted (N. Smolr nd B.E. Griffin, personl communiction). The deletion in mutnt dl-8 does not pper to lter the tryptic fingerprint of lrge T ntigen (Fig ), ut it lters the fingerprint of middle T ntigen (Fig. 3). There is only one methionine-contining tryptic peptide in ech sequence expected to e ffected y the deletion in dl-3. However, in oth cses, the size of the methionine tryptic peptide is very lrge (59 nd 5 mino cid residues for lrge nd middle T ntigen, respectively). Due to the insoluility of MIDDLE T ANTIGEN OF POLYOMA VIRUS 9 TABLE. Plting efficiencies of dl-8- nd dl-3- trnsformed cells in soft gr' (No. of colonies/no. of Cells cells seeded) x 100 (%) 9 dys 17 dys wt-t 77 wt-y 61 wt-z 65 dl-8-t dl-8-y 6 dl-8-z 71 dl-3- () (13.) dl-3-3 (11) (40.0) dl-3-1 (1.7) (4.8) dl-3-13 (9) (43.0) Nontrnsformed cells (<0.) Cells (5 x 103 or 5 x 104) were suspended in 0.3% gr nd incuted t 37 C. Visile colonies ppered y 9 dys in the cse of dl-8- nd wild type-trnsformed cells. The numers of colonies were counted, nd the efficiency of plting ws clculted. In the cse of dl-3-trnsformed ceus, very smll visile colonies ppered only fter further week of incution. Slowly growing colonies were counted under the microscope 9 nd 17 dys fter plting, nd the plting efficiencies re given in prentheses. Nontrnsformed cells were lso exmined under the microscope fter 17 dys. such lrge molecules in solvents used in the current experiments, these peptides proly do not pper on the fingerprints in Fig. nd 3. The results from the peptide nlyses descried here re consistent with the prediction from DNA sequence dt on the choice of the reding frmes in the overlpping region for the two proteins (33). It hs een reported tht virl RNAs present in Py6 cells lck some sequences within the restriction enzyme frgment HpII- (1). More recent results indicte tht the RNA trnscripts of the cells lck the sequence eyond out mp unit 15 (R. Kmen, personl communiction). Therefore, the results shown in Fig. 4A nd 5 suggest tht the position of the croxy terminus of middle T ntigen does not exceed out mp position 15. Peptide mpping dt, together with the nucleotide sequence dt of virl DNA (33) nd nlysis of spliced RNA of polyom virus (R. Kmen, J. Fvloro, J. Prker, R. Treismn, L. Lni, M. Fried, nd A. Mellor, Cold Spring Hror Symp. Qunt. Biol., in press), llow us to propose tht the most likely coding regions for the three species of polyom virus T ntigens re those shown in Fig. 9. By improving the detection procedure (17), we were le to study polyom virus T ntigens in lrge numer of trnsformed cells. Fifty

12 30 ITO, SPURR, AND GRIFFIN J. VIROL. EARLY REGION OF POLYOMA VIRUS GENOME MAP UNITS BASE PAIRS CODING FRAMES LARGE T MIDDLE T SMALL T MUTANTS Hpll 7, / IlflhllllllllEl 1p I1-1 I I N"i " H 11il1 I sl//// I I 3 Hi NIi ii i i " i i W11111ii I ii IN H i i N N N = NG18 dl-8 dl-3 ts A FIG. 9. Possile coding regions nd frmes for lrge, middle, nd smll T ntigens. Short verticl lines in the coding frmes 1,, nd 3 indicte the positions of termintion codons tken from Soed et l. (33). The positions of the deletions of NG-18, dl-8, nd dl-3 nd the region where tsa mutnts mp re indicted. See Griffin et l. (11) for mp units. independently otined lines of cells from rt, hmster, nd mouse origin were exmined for the T-ntigen species present in the cells. The results show tht every line of polyom virustrnsformed cells tested in this study contined middle nd smll T ntigens of the size predicted. Lrge T ntigen, on the other hnd, ws present only in out two-thirds of the lines tested. Other lines of cells either contined no detectle lrge T ntigen or contined truncted forms of this protein which retined the mino terminus ut lcked vrying mounts of mino cids from the croxy-terminl region of the molecule. In two cses exmined, proteins greter in size thn lrge T ntigen (117,000 nd over 150,000 dltons) were present. It hs een shown tht polyom virus-trnsformed rt cells often contin free (nonintegrted) s well s integrted virl DNA (3, 35). There is no wy of knowing in the present study whether the proteins detected re directed from the integrted virl genome or from tht present in free form. It is possile tht protein products from oth virl forms re eing oserved. In the cse of trnsformed mouse cells, however, it is known tht free virl DNA is usully sent. The generl protein pttern of mouse cells, however, does not pper to e unique compred with those of rt nd hmster cells, in the sense tht in ll cses middle nd smll T ntigens re present ut lrge T ntigen my e undetectle or vry in oth size nd mount. Therefore, it seems resonle to ssume tht the integrtion of virl DNA into the host DNA occurs in such wy s to preserve in uninterrupted fshion the entire coding sequence for middle nd smll T ntigens nd tht the norml sizes of lrge T ntigens seen so frequently result from the "disturnce" of the coding sequence for the lrge T unique region y the joining of virl DNA to the host cell DNA within tht region (see Fig. 9). This is entirely consistent with the erlier reports tht virl RNA present in severl lines of trnsformed mouse cells seems to lck some sequences from 3' end of the erly RNA molecules (1, 1). One of the immedite consequences of this is tht integrted virl DNAs which lck the sequences encoding the croxyterminl regions of lrge T ntigen my not hve the norml signls for termintion of trnsltion, polydenyltion, or termintion of trnscription which, in the cse of productive infection, re usully used. Therefore, there is possiility tht virl RNA present in trnsformed cells my e processed premturely or tht trnscription my pss through the virus-host junction nd processing signls present within host cell DNA my e used. If so, vrile sized lrge T-relted proteins would e generted, nd some of these could e unusully lrge. This interprettion itself leves open the possiility tht the joining of virl nd cellulr DNA might occur t specific site in the virl DNA nd t vrile sites in cellulr DNA. Our dt, however, ex-

13 VOL. 35, 1980 clude this possiility, since the numer of peptides unique to lrge T present in truncted lrge T decreses with the size of the protein. Therefore, the integrtion of virl DNA, when it occurs within the distl hlf of the erly region, occurs t more thn one site. In the cse of cells which do not contin detectle mount of lrge T- relted protein, RNA trnscripts for puttive truncted lrge T ntigen my lck one of these processing signls. Truncted lrge T ntigens of less thn out 45K might e directed y other inserts of virl DNA or, lterntively, my result from unstle lrger proteins. The possile trnsltion of proteins from mrna's trnscried from integrted s well s free virl genomes, the integrtion of virl DNA t different sites in the virl s well s the cellulr DNA, nd multiple inserts of virl DNA could explin why the generl T-ntigen pttern of polyom virus-trnsformed cells is so complex. One possile explntion for the oservtion tht oth middle nd smll T ntigens re found universlly in trnsformed cells is tht some coding region for middle T ntigen might lso encode n essentil function for the expression of smll T ntigen. For exmple, it hs een found tht the sequence AATAAA, which my e signl for polydenyltion of eucryotic mrna's, is present in the virl DNA t out mp unit 99 s well s t out 6 (33). If the sequence t mp unit 99 serves s the signl for the polydenyltion of virl mrna's in trnsformed cells, then middle T ntigen could e present in trnsformed cells regrdless of its function. However, results from the studies of the T ntigens of the mutnts dl-8 nd dl-3 fvor the interprettion tht the functionl middle T, rther thn smll T, is indispensle for the trnsformtion mintennce function. In spite of the fct tht intct smll T is present in dl-3-trnsformed cells, only smll colonies re formed in semisolid medium nd smll foci re formed in monolyers on the plstic surfce in liquid medium. In dl-8-trnsformed cells, col- MIDDLE T ANTIGEN OF POLYOMA VIRUS 31 onies nd foci re lrger, on the verge, thn those induced y wild-type virus in spite of the fct tht the smll T ntigen of the mutnt is norml. Although the results certinly do not exclude the possiility tht smll T ntigen my lso ply some role in trnsformtion, they suggest tht one or oth of the two gene products (lrge or middle T ntigens or oth) which re ffected y the dl-8 nd dl-3 muttions re primrily responsile for inducing the full expression of the phenotype of trnsformtion. As indicted in Tle 1, the screening experiments included cells trnsformed y the dl-8 mutnt, which showed exctly the sme kinds of constncy nd of vrition s the wild typetrnsformed cells. dl-3-trnsformed cells were lso found to e the sme in this respect, lthough the numer of cell lines exmined so fr is smll. In other words, irrespective of the size or the mount of lrge T-relted protein in the cells, chrcteristics of the phenotype of the cells were entirely dependent on the mutnt used. This implies tht middle T ntigen plys custive role in inducing the trnsformtion phenotype. The simplest interprettion of the results with the mutnts is tht dl-8 nd dl-3 re mintennce-type mutnts nd tht the ltered middle T proteins of dl-8 or dl-3 hve "stronger" or "weker" ctivities, respectively, for inducing the trnsformtion phenotype. One of the remining questions to e nswered is why some lines of cells contin lrge T ntigen nd others do not. Some lines listed in Tle 1 contin no unintegrted virl DNA (F. Birg nd R. Kmen, personl communiction). At lest one of them contins lrge T ntigen (Y. Ito, mnuscript in preprtion). Whether or not this lrge T ntigen is iologiclly functionl is currently under investigtion. LITERATURE CITED 1. Bcheler, L. T Virus-specific trnscription in 3T3 cells trnsformed y the ts- mutnt of polyom virus. J. Virol. : Benjmin, T. L Host rnge mutnts of polyom virus. Proc. Ntl. Acd. Sci. U.S.A. 67: Birg, F., R. Dulecco, M. Fried, nd R. Kmen Stte nd orgniztion of polyom virus DNA sequences in trnsformed rt cell lines. J. Virol. 9: Crwford, L. V., C. N. Cole, A. E. Smith, E. Puch, P. Tegtmeyer, K. Rundell, nd P. Berg Orgniztion nd expression of erly genes of Simin virus 40. Proc. Ntl. Acd. Sci. U.S.A. 75: Dvidson, E. A., nd I. McPherson Synthesis of complex scchrides y synchronised NIL-8 hmster cells. Exp. Cell Res. 95: Eckhrt, W Complementtion etween temperture sensitive nd host rnge non-trnsforming mutnts of polyom virus. Virology 77: Fluck, M. M., R. J. Stneloni, nd T. L. Benjmin Hr-t nd ts-: two erly gene functions of polyom virus. Virology 77: Freemn, A. E., R. V. Gilden, M. L. Vernon, R. G. Wolford, P. E. Hugnin, nd R. J. Huener Bromo- deoxyuridine potentition of trnsformtion of rt emryo cells induced in vitro y 3-methyl-cholnthrne: induction of rt leukemi virus gs ntigen in trnsformed cells. Proc. Ntl. Acd. Sci. U.S.A. 70: Fried, M Cell trnsformtion ility of temperture-sensitive mutnt of polyom virus. Proc. Ntl. Acd. Sci. U.S.A. 53: Fried, M., B. E. Griffin, E. Lund, nd D. L. Roerson Polyom virus-a study of wild-type, mutnt nd defective DNAs. Cold Spring Hror Symp. Qunt. Biol. 39: Griffin, B. E., M. Fried, nd A. Cowie Polyom DNA: physicl mp. Proc. Ntl. Acd. Sci. U.S.A. 71: Griffin, B. E., nd C. Mddock New clsses of

14 3 ITO, SPURR, AND GRIFFIN vile deletion mutnts in the erly region of polyom virus. J. Virol. 31: Httori, J., G. G. Crmichel, nd T. L. Benjmin DNA sequence ltertions in Hr-t deletion mutnts of polyom virus. Cell 16: Hunter, T., M. A. Hutchinson, nd W. Eckhrt Trnsltion of polyom virus T-ntigens in vitro. Proc. Ntl. Acd. Sci. U.S.A. 75: Hutchinson, M. A., T. Hunter, nd W. Eckhrt Chrcteriztion of T ntigens in polyom-infected nd trnsformed cells. Cell 15: Isrel, M. A., D. T. Simmons, S. Hourihn, W. P. Rowe, nd M. A. Mrtin Interrupting the erly region of the polyom virus DNA enhnces tumorigenicity. Proc. Ntl. Acd. Sci. U.S.A. 76: Ito, Y Polyom virus-specific 55K protein isolted from plsm memrne of productively infected cells is virus-coded nd importnt for cell trnsformtion. Virology 98: Ito, Y., J. R. Brocklehurst, nd R. Dulecco Virus-specific proteins in the plsm memrne of cells lyticlly infected or trnsformed y polyom virus. Proc. Ntl. Acd. Sci. U.S.A. 74: Ito, Y., J. R. Brocklehurst, N. Spurr, M. Griffiths, J. Hurst, nd M. Fried Polyom virus wild type nd mutnt T-ntigens in erly proteins of oncogenic viruses. INSERM Colloq. 69: Ito, Y., N. Spurr, nd R. Dulecco Chrcteriztion of polyom virus T ntigen. Proc. Ntl. Acd. Sci. U.S.A. 74: Kmen, R., D. M. Lindstrom, H. Shure, nd R. W. Old Virus specific RNA in cells productively infected or trnsformed y polyom virus. Cold Spring Hror Symp. Qunt. Biol. 39: Kimur, G Temperture-sensitive growth of cells trnsformed y ts- mutnt of polyom virus. Nture (London) 153: Kimur, G., A. Itgki, nd J. Summers Rt cell line 3YI nd its virogenic polyom nd SV40-trnsformed derivtives. Int. J. Cncer 15: Lni, L., M. Griffiths, B. Cooke, Y. Ito, nd M. Fried Untrnsformed rt cells contining free nd integrted DNA of polyom non-trnsforming (hr-t) mutnt. Cell 19: J. VIROL. 5. McPherson, I., nd L. Montgnier Agr suspension culture for selective ssy of cells trnsformed y polyom virus. Virology 3: Schffhusen, B. S., J. E. Silver, nd T. Benjmin Tumour ntigen(s) in cells productively infected y wild-type polyom virus nd mutnt NG-18. Proc. Ntl. Acd. Sci. U.S.A. 75: Schlegel, R., nd T. Benjmin Cellulr ltertions dependent upon the polyom virus hr-t function: seprtion of mitogenic from trnsforming cpcities. Cell 14: Seif, R., nd F. Cuzin Temperture-sensitive growth regultion in one type of trnsformed rt cells induced y the ts- mutnt of polyom virus. J. Virol. 4: Simmons, D. T., nd M. A. Mrtin Common methionine-tryptic peptides ner the mino-terminl end of primte ppovvirus tumor ntigens. Proc. Ntl. Acd. Sci. U.S.A. 75: Simmons, D. T., C. Chng, nd M. A. Mrtin Multiple forms of polyom virus tumor ntigens from infected nd trnsformed cells. J. Virol. 9: Smrt, J. E., nd Y. Ito Three species of polyom virus tumor ntigens shre common peptides proly ner the mino termini of the proteins. Cell 15: Soed, E., J. R. Arrnd, N. Smolr, nd B. E. Griffin Sequence from the erly region of polyom virus DNA tht contins the origin of repliction nd codes for smll, middle nd (prt of) lrge T ntigens. Cell 17: Soed, E., J. R. Arrnd, N. Smolr, J. E. Wlsh, nd B. E. Griffin Coding potentil nd regultory signls of smll DNA tumor viruses s suggested y the sequence of the polyom virus genome. Nture (London) 83: Soed, E., nd B. E Griffin Sequences from the genome of non-trnsforming mutnt of polyom virus. Nture (London) 76: Zouzis, D., I. Prsd, nd C. Bsilico Stte of the virl DNA in rt cells trnsformed y polyom virus. II. Identifiction of the cells contining nonintegrted virl DNA nd the effect of virl muttions. J. Virol. 4: