Polyoma virus proteins

VIROLOGY

46, 555-5613

(1971)

Polyoma

Virus

Proteins

I. Multiple

Virion

Components

R. ROBLIN,’ Salk

Institute

for

E. HARLE,” Biological

AND

Studies,

Accepted

April

San

R. DULBECCO Diego,

California

9211$

16, 1971

The polypeptide components of highly purified polyoma virions were separated by electrophoresis on SDS-polyacrylamide gels. Seven distinct virion polypeptides were reproducibly observed, and at least six of these components are unique polypeptides, not aggregates. The sum of the molecular weight.s estimated for these virion polypeptides nearly equals or exceeds the coding capacity of the viral DNA. The major capsid protein, P2, contains 5&70’3$, of the radioactivity and has an estimated molecular weight of 48,000 daltons. Three small (MW 15,000-19,000) polypeptides, which have a high lyaine to valine ratio, are apparently “internal” proteins with an affinity for viral DNA. Several possible explanations for the large number of minor virion polypeptides are discussed.

gested the existence of one major protein component (Thorne and Warden, 1967; Kass, 1970), or one major component> plus one “internal” protein component (Fine et al., 1968) in polyoma virus particles. In contrast to these earlier reports, we find t,hat purified polyoma virus preparations, when examined by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels, contain at least six different polypeptide components. We have also determined approximate molecular weights for each of t)hese component’s, and considered some possible explanations for the origin of these multiple protein components.

INTRODUCTION

Cell transformation in vitro by the small DD;A tumor viruses, polyoma and SV40, is being intensively studied as a simple experimental carcinogenic system (Dulbecco, 1969). The small DNA genomes of polyoma (Follett and Crawford, 1968) and SV40 (Anderer et al., 1967) are estimated to contain sufficient genetic informat’ion to code for only (i-10 proteins of 20,000 daltons molecular weight (MW). Since the activity of one or more virus genes appears to be required to maintain certain aspects of the phenot,ype of the transformed cell (Eckhart et al., 1971), identification and characterization of polyoma virus specific proteins is an important, first, step for research on the mechanism of polyoma virus-induced cell transformation. To begin characterization of polyoma virus-specific proteins, we have reexamined the polypeptide components found in purified virus particles. Previous work has sug-

MATERIALS

AN11

METHODS

Virus strains. A large-plaque strain of polyoma virus (Vogt, and Dulbecco, 1962) was obtained t,hrough t’he courtesy of Dr. Marguerite Vogt. A single virus pool, prepared by infection of primary baby mouse kidney (BRIK) cell cultures at an input multiplicit,y of about 20 PFU/cell, which titered 8 X lo8 PFUiml, was used in most of the experiments reported here. Some experiments indicated below utilized a virus pool derived from large-plaque virus

1 Present address: Infectious Disease Unit, Massachusetts General Hospital, Boston, Massachuset,ts 02114. 2 Present address: Genetisches Institut der Universitaet, 73 Fraiburg, Schaenzlestr. 11, Germany. 555

556

ROBLIN,

HitRLE,

by two cycles of plaque purification in secondary mouse embryo cells. The final “plaque purified” virus pool was prepared by infection of primary BMK cell cultures (input multiplicity about 0.01 PFU/cell) and had a titer of 4 X log PFU/ml. Although they are not described in any detail below, some additional experiments have been done with a polyoma virus strain designated “small plaque” obtained from Dr. William Murakami. A pool of this virus was similarly prepared by infection of primary BMK cell cultures at a multiplicity of about 10 PFU/cell. We observed no qualitative difference in the protein pattern on SDS gels between this strain and the large-plaque strain used in all our experiments. Bufers. Tris-buffered saline (TBS) contains, per liter, MgC12.6Hg0, 0.1 g; CaClz , 0.1 g; NaCl, 8 g; KCl, 0.38 g; Na2HP04, 0.1 g; Sigma 7-9, 3.0 g. TBS buffer minus magnesium and calcium is designated TD buffer. Growth and labeling of virus. Radioactive polyoma virus was prepared by infection of confluent primary BMK cultures with input multiplicities of lo-50 PFU/cell. After adsorption of the inoculum (0.4 ml in TBS/SO mm petri dish) for 1 hr at 37”, Dulbecco’s modified Eagle’s medium (DEM) was added. DEM contains four times the usual concentration (Eagle, 1959) of amino acids and vitamins, plus 500 U/ml penicillin, 100 pg/ ml streptomycin, and 10 U/ml mycostatin. Medium added to the cultures after infection did not contain serum, since it was found that serum omission had no measurable effect on virus yield. At 16-24 hr after infection, this medium was removed and replaced with DEM containing one-tenth the above amounts of leutine, lysine, and valine, and the desired amounts of radioactive amino acids. Reduction of the concentrations of leucine, lysine, and valine to one-tenth their usual concentration in DEM had no appreciable effect on the virus yield, measured by hemagglutination at 72-96 hr post infection. To label polyoma virus proteins with arginine, citrulline (final concentration 0.4 mM) was added to the growth medium in place of arginine. Substitution of citrulline for argi-

AND

DULBECCO

nine also had no detectable effect on virus yield. To label the virus proteins with cysteine or tryptophan, cystine-35S (6.25 &i/ ml medium) or tryptophan-3H (10 &X/ml medium) was added directly to DEM. In a typical labeling experiment, incorporation of leucine-3H (2 &X/ml medium), lysine-3H (2 #X/ml medium) and valineJH (2 &i/ml medium) yielded purified virions with a specific activity of about 3000 cpm/pg protein (as bovine serum albumin by Lowry protein assay). The radioactivity was measured on a 20+1 aliquot of virus in 10 ml of Bray’s scintillation solution using a Beckman scintillation counter. Virus purijication. At 72-96 hr after infection, when the cells exhibit an almost complete cytopathic effect, the cultures were frozen at -20°C. The frozen cultures were thawed in an incubator at 35-37”, then the medium was withdrawn and centrifuged at low speed (International centrifuge, 500 ~1, 15 min at 4’) to remove cell debris. Portions, 26 ml, of the supernatant fluid were carefully layered atop 10 ml saturated KBr solution (0.05 M Tris, pH 8, 0.01 M EDTA) and centrifuged in the Spinco SW 27 rotor at 20,000 rpm for 3 hr at 20”. (Alternatively, 22-ml portions of the supernatant fluid were layered atop 8 ml of saturated KBr solution and centrifuged at 23,000 rpm in the Spinco SW 25.1 rotor for 3 hr at 20”). Viral material formed two sharp visible bands under these conditions. Both bands were collected and dialyzed against at least two changes of 500 volumes of TD buffer. Portions (3.6 ml) of dialyzed KBr virus fraction were mixed with 1.7 ml saturated CsCl solution (in TD buffer) and centrifuged in the Spinco angle head 40 rotor at 38,000 rpm at 20” for 60 hr. The virus material now formed t’wo or more well separated bands which were collected separately by puncturing the bottom of t’he t,ube. Fractions of the CsCl gradient containing the virus peak (i.e., the lower band) were pooled, dialyzed against at least two changes of 1000 volumes of 0.01 M phosphate buffer, pH 7.2, and stored at 4”. Virus and hemagglutination assays. Polyoma virus infectivity was assayed by plaque formation on secondary mouse embryo cell cultures (Crawford et al., 1962). The dilut’ed

POLYOMA

VIRION

virus suspension (0.2 ml/60 mm petri dish) was adsorbed for 1 hr at’ 37”, then t,he plates were overlaid with DE&l medium containing .j ‘b horse serum and 0.9 % agar. The plates were usually incubat,ed at 33” rather t)han 37” because the plaques were more distinct and easier to score at the lower temperature. ;1 second agar overlay (DERI medium, 5 % horse serum, 0.9 %. agar plus 0.005 % neutral red) was added to the plates 9-10 days after infection, and the plaques were scored 14-21 days after infection. Hemagglutination was assayed by combining serial 2-fold dilutions of virus (0.4 ml in TD bufferj wi-ith 0.4 ml guinea pig red blood cells (5 X 10’ cells/ml). After incubation at, 37” for 30 min, the tubes were shaken and allowed to set,tle at 4”. The last dilution which showed complete agglutination was taken as t,he end point’. The reciprocal of the end point dilut’ion equals t,he hemagglutination titer (expressed as unit’s/ml). T’i,us disrupfion, recluction and allcylation cd‘ kal proteins. When necessary, CsClpurified virus was dialyzed against, 0.01 M phosphate buffer pH, 7.2, and concentrated about lo-fold to approximately 0.1 ml by rotary evaporation under vacuum at 37”. C’oncentrntion by this t,echnique produced no change in the sedimentation pattern of t,he virus on sucrose gradients. Purified virus preparat.ions in 0.01-0.1 A1 phosphate buffer, pH 7.2, were disrupted by adding SDS, dit’hiothreitol, and Tris buffer (pH S.5) to final concentrations of l%, 0.01 31, and 0.1 U, respectively (result,ant pH = S&&4). The solution w-as t’hen heluted in a boiling wat#er bath for 3 min. After t,he solution had cooled, one-fifth volume of iodoacet,amide (1.2 A1 in 1 N Tris buffer, pH 5.5) was added and the solution was allowed t’o stand at 20”-25” for 2-4 hr. The reduced, alkylated proteins were then dialyzed against 1000 volumes of gel electrophoresis sample buffer (0.1% SDS, 0.01 J1 phosphate buffer, pH 7.2) for 12-18 hr. l’olyacrylamide gel electrophoresis. Acryla,mide gels of varying lengths (usually 7.5 or 15 cmj and varying acrylamide concentrations (7.5, 10, or 15%) mere prepared, polymerized, and run essentially as described by Ylaizel (1969). The gel buffer contained

--c ;).)I

PROTEINS

0.1% SDS, 0.05 M phosphate buffer, pH 7.2, as did the tray buffer. One-sixth volume of 60 % sucrose was added t’o each sample before layering it on the gel. Optimal resolution of the virion polypeptides (especially components 1’5-7) was obtained when a small sample volumes (less than 0.13 ml) \\-erc electrophoresed on long (15 cm) gels cont,aining 15 %l acrylamide. Gel jractionation and counting. Radioactive gels were frozen on dry ice and sliced by hand using a cutt’er cont,aining a series of par:dlel razor blades. Each 1.0-1.2 mm slice \vas combined wit,11 0.1 ml 30 ‘X hydrogen peroxide in a glass scintillation vial, and heated at SO”-60” for 4-6 hr, until the slice dissolved. When the vials had cooled, 10 ml Bray’s scint.i11:1tion solut’ion was added and t,he radioactivit? was determined in :I Beckman scint,illat)ion counter. Radioactive standards, for corrw t,ion of 3H and 14C cpm for spillover, wre prepared by adding 5-~1 amounts of “H or 14C to samples from t,he gel which contained only background levels of radioa&\-it,y and then recounting these samples. Ch.enzicals. The following products were obtained from the sources designated. l’encillin, streptomycin, and mvcostatin \vere obtained from Squibb. Leucine-RH (40 Ci mmole), l\Gne-3H (50.4 Ci/mmole), valinr3H (0.6 C’i,/mmole), and arginine-aH (1s Ci mmole), valine-14C (260 mCi/mmole), t ryptophan-“H (6.5 Ci/mmole), nrginine-‘4C (:31:! mCi,/mmole), cystine-““S (27.5 mCi/mmole), and thgmidineJH were products of Sch~lrz BioResearch. Iodoacetamide (Jl.ll. grade) and guanidine hydrochloride (Ultra Pure) were obtained from ~Iann, and dithiothreitol (A grade) from Calbiochem. IWSUI,TP Virus Purification Sedimentation onto a cushion of saturated RBr solution (Mattern et al., 1967) proved to be an effective technique for initial concentration of radioactive polyoma virus from crude lysates and for separation of the virus from the bulk of contaminating cellular proteins. Figure 1 ~110~s the results of a mixing experiment, designed to test8 whet,her labeled cellular proteins were efficientsly rcmoved from virions by the ICBr and C&l

558

BOBLIN, KS,

HARLE,

GRADIENT

I

FRACTION

NUMBER

FIG. 1. Mixing experiment to test virus purification technique. Mock-infected primary BMK cells, labeled with a mixture of leucine-3H, lysineBH, valine-3H, and arginine-aH from 25 to 75 hr after mock-infection, were frozen and thawed once; 80 ml of the supernatant fluid was mixed with 30 ml of supernatant fluid from once frozen and thawed primary BMK cells, which had been infected with plaque-purified polyoma virus stock and labeled with lysine-14C (0.5&i/ml of medium) from 25 to 75 hours post infection. Virus was then purified from this mixture as described under Materials and Methods. Aliquots of the XBr and CsCl fractions were precipitated with ice cold 10% trichloroacetic acid (TCA), filtered onto glass fiber filters, dried, and counted in toluene scintillator under conditions permitting accurate separation of 3H and 14C counts per minute. Bottom third of KBr Gradient: Fractions of 0.37 ml each were collected and 204 aliquots were precipitated and counted. Arrow from “1°C Infected” points to one of the two visible bands of virus material. Bar indicates the fractions which were pooled, dialyzed, and centrifuged to equilibrium in CsCl. CsCZ Gradient: Fractions of 0.16 ml each were collected, and 50-J aliquots were precipitated and counted. A second 50.~1 aliquot was combined with 0.35 ml of TD buffer and assayed for hemagglutinating activity as described in Materials and Methods.

gradient centrifugations. Mock-infected primary BMK cells labeled with a mixture of 3H-amino acids were mixed with a lysate of polyoma virus-infected BMK cells labeled with lysineJ4C and the virus was purified as described in Materials and Methods. Although some 3H-radioactivity from mockinfected cells still contaminated the virus bands after the KBr gradient (Fig. l), most

AND

DULBECCO

of the 3H-radioactivity does not sediment, as far into the KBr gradient as do t)he virus bands. Subsequent CsCl equilibrium cemrifugation in an angle rotor essentially completely separated the remaining contaminating 3H-radioactivity from the virus peak region (fractions 10-15). This experiment demonstrates that any labeled cellular proteins from mock-infected cells which might stick to polyoma virions are removed by the high salt conditions of the KBr and CsCl gradients. However, the mixing experiment cannot exclude the more remote possibility that some other, different cellular proteins, whose synthesis is induced or stimulated in polyoma-infected BMK cells, might bind to the virions and be carried through the purification process as contaminants. Separate experiments established that I520 % of the plaque-forming unit’s (PFU) present in the crude lysate were recovered in the lower, virus-containing band after CsCl centrifugation. Thus, some inactivation of polyoma virus infectivity may take place during this purification procedure. Two other peaks of hemagglutinating activity were detected in fractions of the CsCl gradients. The heavier of these components (fractions 21-23, Fig. 1) may correspond t,o the “light pseudovirions” previously observed by Michel et al., 1967). In addition, this region of the CsCl gradient contains much material which has the appearance of separated virus capsomereswhen examined in the electron microscope. Fractions from this region usually aggregate and precipitate upon removal of the CsCl by dialysis. The lightest component (fractions 26-28 of the CsCl gradient) appeared to consist mostly of empty virus particles (L’shells”), since the particles were completely penetrated by uranyl acetate when negatively stained preparations were examined by electron microscopy and since the particles sedimented more slowly than virions upon sucrose gradient centrifugation. The relative amounts of these two hemagglutinating components varied considerably from one virus preparation to another. Sucrose gradient centrifugation of the virus peak from CsCl gradients showed SO-

POLYOMA

VIRION

90 % of the radioactivity sedimenting as a single sharp peak (Fig. 2). Electron microscope examination of pools of the CsCl virus peak showed particles with the size and morphology expected for polyoma virus (Fig. 3). Since the SDS-polyacrylamide gel pattern of virus samples aft’er sucrose gradient sedimentat,ion was qualitatively and quantitatively identical to that of virus purified only t’hrough the C&l gradient step, all experiments described used CsCl purified virions. SDS Gel E’lectrophoresis When purified samples of polyoma virus labeled with radioactive amino acids were disrupted in 1% SDS solution, reduced and alkylated, and then run on SDS-polyacrylamide gels, seven peaks of radioactivity (PlP7) were observed (Fig. 4). Qualitatively, this pattern of one major peak and six minor peaks has been observed in more than twenty different virus preparations labeled with different combinations of radioactive amino acids. When virions labeled with thymidine3H and amino acids-‘*C were disrupted and electrophoresed-on SDS gels, all the 3H-cpm were found to be located in the first three slices from the top of the gel. Thus, the viral DNA apparently does not migrate into the gel, and a complet’e separation of viral pro-

FIG. 2. Sucrose gradient centrifugation of CsCl purified virus. Five to 20% sucrose gradients (0.15 M NaCl, 0.05 M Tris pH 8, 0.01 M EDTA buffer) were formed over a 1 ml cushion of C&l solution (density, 1.58 g/ml) in Tn. Plaque-purified polyoma virus after CsCl purification (25 wg protein in 0.15 ml TD buffer) was layered atop the gadient and the t,ube was centrifuged in the Spinco SW 41 rot,or at 20,000 rpm and 20” for 90 mins. Fractions of 15 drops were collected and counted directly in 10 ml of Bray’s scintillation solution.

.-.!I!)

PROTEINS

teins from viral DNA is eff ect,ed by the virus disruption conditions. When virus from a mixing experiment similar to that described in Fig. 1 was examined by SDS-gel electrophoresis (Fig. 4)) no 3H-radioactivity from mock-infected cells was found at the positions of any of the minor components. The distribution of radioactivity among the different polypeptide peaks for three independently grown and purified polyoma virus preparations labeled with arginine or lysine is shown in Table 1. These results indicate t>hat the relative proportions of the seven polypeptide components are quite reproducible. Reduction and alkylation of the virus proteins was found t,o be required to prevent variable amounts of aggregation which led to the appearance of additional peaks between the top of the gel and component P2. Six Distinct

Polypeptide

Components

SDS-gel electrophoresis of virus preparations grown in the presence of different combinations of 3H- and ‘*C-labeled amino acids provided evidence t’hat at, least six of the seven peaks were distinctly different polypeptides and not aggregates. Both qualitative and quantitative differences were observed between the different peaks. For example, components P5, P6, and 1’7 were found to contain no t,ryptophan in a double labeling experiment utilizing t,ryptophan-3H and arginine-‘*C (Fig. 5). In other tlxperiments, in which the virus was labeled by growt’h in t’he presence of cystine-3”S and arginine-“H t’hen analyzed by SDS-gel electrophoresis, cysteine label could be detected only in components Pl, P2, and P.i. Differences between the seven polypeptide components of polyoma virions are summarized in Table 2. Taken together, these results indicate t,hat at least six of the seven polypeptide components observed in polyoma virions are dist’inctly different, and are not thcl result of aggregat,ion. The only possible aggregate is component’ PI. This component is cluantit,atively the most variable component (Table l), and its moleculw weight and other properties (Table 2) do not, yet eliminate the possibility that, component, 1’1 is :L clitnclr of

ROBLIN,

HiLRLE,

AND DULBECCO

FIG. 3. CsCl-purified polyoma virions. A drop of CsCl-purified polyoma virions (0.17 mg protein/ml in TD buffer) was placed for 1 min on a 300.mesh copper grid covered with a carbon on nitrocellulose film, and then removed with filter paper. The grid was then washed three times with drops of distilled water, followed by 3 drops of 2% uranyl acetate, pH 4.5. After drying, it was examined in a Hitachi Model 11B operated at 75 kV. The sedimentation pattern of an aliquot of this same virus pool is shown in Fig. 2. Scale line indicates 1000 A.

were separated by SDS-gel electrophoresis, then eluted from the gel, concentjrated, and run OIL a second SDS-gel in the presence of 3H-labeled polyoma virus prot,eins as markers. The 14C-labeled eluted proteins (cont,aining components 1’4 and 1’5 -7) were found to migrate identically to the :‘)Ilabeled marker proteins (Pig. 6). In pmt,icu lar, the smaller 14C-l:Lbeled elut#ed proteins showed no tendency to aggregut e to t,he higher molecular weight components PI and P2. Similar result,s have been obtained for components 1’2 and 1’3.

.

/ 0

10

20 SLICE

30

40

50

60

NUMBER

PIG. 4. SDS-acrylamide gel of polyoma virion prot,eins. A large-plaque polyoma virus sample was reduced with 0.01 M P-mercaptoet,hanol and alkylated as described under Materials and Methods. The sample was electrophoresed on a 7.5 cm gel containing 7.570 acrylamide in 0.170 SDS, 0.05 M phosphate buffer, pH 7.2 for 2.5 hr at 10 mA/gel. The gel was fixed overnight in 12.5% TCA, then sliced, dissolved and counted as described in Materials and Methods. The virus sample was from a mixing experiment, similar to that described in Fig. 1, in which t,he infected cells were labeled with leucine-14C (0.18 &i/ml medium, 25-96 hr after infection) and the mock-infected cells were labeled with leucine-3H, lysine-“H, and valine-3H (4.5 &i total/ml medium, 25-96 hr after mock-infection). The tiny peak of “H-counts per minute under peak P2 probably is the result of inaccurate correction for spillover into the 3H-channel. Ninety-five percent of t)he radioactivity applied to tho gel was recovered.

component, 1’2. If, however, Pl is a noncovalently bonded dimer of P2, it is unusually resistant’ to denaturing agents, since t,he amount of Pl is minimized but not eliminated if the virus is reduced and alkylated in 6 31 guanidine hydrochloride at 37”. Comparison of the pept)ides liberated by trypsin from separated components Pl and P2 is being undertaken to resolve this question. Additional evidence that none of t#he other components are aggregates was obtained when t4Clabeled polyoma virus proteins

Approximate molecular weights of proteins can be determined from their relat,ive migration in SDS gels (Shapiro et al., 1967; Weber and &born, 1969; Dunker and lteuckert ,1969). The molecular weights of polyoma virus prot,eins were estimated both by coelect,rophoresis with differentially labeled +X174 bact.eriophage coat protJeins (Burgess, 1969) and by electrophoresis in the presence of prot,ein markers of known molecular weight. Similar results were obtained by both met’hods. Estimates of t,he molecula,r weights of t’he different, polyoma protein components are summarized in Table 2.

Polyoma virus preparations grown in mouse kidney cells have previously been shown t,o contain pieces of cellular DNA surrounded by virus capdd protein or “pseudovirions” (illichel ef al., 1967; Winocour, 196X). Thus, some of the minor polyoma protein components might be present) in purified virion preparations because they bind to cellular DNA in pseudovirions. Of particular interest, in this respect are componems 1’5, P6, and 1’7 which other experiments (P’ine and Murakami, personal communication, 1970; Roblin, unpublished data) have shown to be missing or much reduced in empty virus “shells” (particles which lack DNA). The ratio of virions to pseudovirions can vary widely from one polyoma preparation to another (Michel et al., 1967). If mby pseudovirions contained protein components P5-7, t!he amount, of components I’57 might be expected to vary from one virus prepara-

562

ROBLIN,

H8RLE,

AND

TABLE QUANTITATIVE

DISTRIBUTION

Percent

Arginine-labeled Preparation 1 2 3 standard

5.7 3.1 3.3 error) (*E)

Lysine-labeled Preparation

Average

4 (virus peak) 5 (heavy side) 5 (light side)

(& standard

error)

5.15 3.10 4.30 -4.18 (*.fw

OR ARGININE

IN POLYOMA

P3

P4

57.5 55.8 63.0 --58.8 (f3.1)

6.8 8.4 6.5

8.1 6.7 8.9

73.31 76.96 75.45 --75.24 kkl.50)

PS-7

7.9 (* .91)

(Z3) 3.26 2.69 3.16

21.9 22.4 18.4 __20.9 (f1.2)

3.34 2.04 2.31 -2.56 (A .56)

3.04 (f.24)

0 Polyoma virions were labeled by growth in the presence of radioactive purified, disrupted, and electrophoresed on SDS-gels as described in Materials

SLICE

VIRION

total cpm in component

P2

Pl

(i

1

OF RADIOACTIVE LYSINE PROTEINS

Virus

Average

DULBECCO

14.94 15.21 14.78 -14.98 (jz.18)

arginine or lysine, then and Methods.

NUMBER

FIG. 5. SDS-acrylamide gel of tryptophan-sH/arginine-1% virus. Large-plaque polyoma virus grown in the presence of tryptophan-8H (10 &i/ml medium) and arginine-14C (0.25pCijml medium) was purified, reduced with 0.01 M &mercaptoethanol and alkylated with iodoacetamide. One hundred seventyfive micrograms of virus protein in 259 ~1 was electrophoresed on a 7.5 cm and 7.5% acrylamide gel at 10 mA/gel for 2.5 hr. The gel was fixed overnight in 12.5% TCA, then sliced, dissolved, and counted as described in Materials and Methods. More than 95% of both the 3H and r*C cpm applied to the gel was recovered.

tion

components,

radioactivity

preparations were examined by SDS-gel electrophoresis. In addition, fractions from the light density side of the virus band in CsCl

to another. Yet, as the data in Table 1 show, the radioactivity in components P5-7 is a rather constant percentage of the total in the separated

virion

protein

when

several

different

virion

POLYOMA 7

1,4BLK Cl< \R.\('TEHIS'PICS

VIltION

.Xi

PKOTISINS

2

OF POLYOMA

VIRION

POLYPEPTIDES

Presence

of

“H :W

Ratio

Com-

ponent

P(i 1’7

bptophan

1 ~ -

cysm teine

i i

-

LYS/ VAT,

VAL/ VAL”

1 .22 1.30 0.66 0.60 2.85 2.3 1 2.1

1.82 1.78 1.71 1.77 1.83 1.77 1.82

-

i

Molecular weight

86,000 48,000 35,000 23,000 19,000 17,000 15,003

u Virus ww grown in the presence (,f lysine-3H (4.4 &i/ml medium) and valine-1% (0.3 &i/ml medium) or valine-3H (5.0 &X/ml medium) and valine-W (0.3 &i/ml medium), then purified, disrupted, and electrophoresed on SDS gels as described in Materials and Methods. The peak ratios of the valine-3H:valine-14C labeled polyoma virion components are included to illustrate the reproducibility of the ratio when the same amino acid is used to label the virus.

gradients can contain an increased proport,ion of pseudovirions (Michel et al., 1967; Winocour, 1968). If polyoma virion components P5-7 were contained only in pseudovirions, then the proportion of these components might be greater in pooled fractions from the light side of the virion band in a CsCl gradient. However, quantitative comparison of t,he proportion of lysine-3H in virion components P5-7 showed no significant difference between pools of virions from the heavy and light sidesof the virus band in CsCl (Table 1, preparation 5). It is possible that virion protein components P5-7 are contained in both virions and pseudovirions, becauseof the identical buoyant density in CsCl of virions and “heavy” pseudovirions. If either only virions or only pseudovirions contained protein components P5--7, t,hen the two types of particles might not be expected t’o have t,he same average bouyant density in CsCl, since protein components P5-7 probably comprise at least 10 %j of the prot’ein content of polyoma virions (Table 1). This argument clearly depends upon the uncertain assumptions that both virion and pseudovirion particles

FIG. 6. Iteelectrophoresis of extracted polyoma proteins. Large- plaque polyoma virus, grown in the presence of leucine-14C, lysine-*%, and valine1% (0.83 &i total/ml medium), was reduced with 0.01 M p-mercaptoethanol and electrophoresed on a gel in 0.1% SDS, 0.1 fil phosphate, pH 7.2. The viral protein peaks, located by slicing and counting an identical “pilot” gel, were eluted from the gel slices with 5 ml of 0.1% SDS, 0.1% P-mercaptoethanol, 0.01 M phosphate buffer. The fractions containing eluted proteins P4 and P5-7 were combined, reduced in volume t,o about 0.1 ml by vacuum evaporation, and dialyzed against electrophoresis sample buffer. This eluted virus protein sample was mixed with a polyoma virus sample labeled with leucine-3H, lysine-3H, and valine3H (6 &i total/ml medium) and electrophoresed on a 7.5 cm 10% acrylamide gel (7.5 mA/gel, 5.8 hr). After overnight fixation in 12.57, TCA, the gel was sliced, dissolved, and counted as described in Materials and Methods.

are equally permeable to cesium ions, and that the presence of components PFj-7 in only one type of particle would not, differentially alter the binding of cesium ions. DISCUSSION

The polypeptides of polyoma virions have been the subject of three previous studies (Thorne and Warden, 1967; Fine et al., 1968; Kass, 1970. It seemslikely that t.hese earlier studies failed to detect the minor components reported here, either becauseof differences in virus preparation or analysis techniques, or because insufficient material

ROBLIN,

HiiRLE,

was analyzed to permit detection of the minor components. The one point upon which all these studies agree is that polyoma virions contain a major protein component which has a molecula,r weight of about 45,000 daltons. It seems likely that this component (P2) is the major protein component of the virus capsid and is virus specific. More recent experiments by Fine and Murakami (personal communication, 1969), utilizing electrophoresis on SDS-gels, have also detected several additional minor polypeptide components which appear to be identical to those described here. Most of the minor components are not observed when polyoma virions are disrupted with 8 M urea and electrophoresed on acrylamide gels in 7-8 M urea (Fine et al., 1968; Kass, 1970; Harle and Roblin, unpublished observations). It may be that treatment of polyoma virions with 8 M urea does not completely dissociate some viral proteins from the viral DNA. Viral proteins remaining attached to the viral DNA would not be expected to migrate into the gel at pH 4.3-4.5. The sum of the molecular weights estimated for components Pl-P7 is about 240,000 daltons (Table 2). Assuming that polyoma viral DNA has a molecular weight of 3 X lo6 daltons, if only one DNA strand is transcribed in vivo, t’hen polyoma virus DNA could code for approximately 170,000 daltons of protein. Thus, the sum of the molecular weights of virion polypeptides nearly equals or exceeds (depending on whether or not PI is included) the estimated coding capacity of the viral DNA. We suggest the following hypotheses to explain this large number of minor protein components. First, some of the minor polyoma virions polypeptide components may be host cell gene products which either are still present as contaminants or actually play a role as structural components of the virus. Although it is not yet possible to rule out completely the presence of “contaminating” host cell proteins, the results of the mixing experiment (Fig. l), failure of further virus purification on sucrose gradients to alter the virion polypeptide composition, and the quantitative reproducibility of the proportion of the minor components in different virus preparations (Table 1) all argue against this interpretation. The possibility that some

AND

DULBECCO

of t’he minor components are host celldirected proteins which also serve as essenGal viral struct)ural components requires further investigation. In particular, the fact that polyoma virions contain three small polypeptide components which are associated with viral DNA and which contain no tryptophan, suggest’sthat these components (P5-7) might be host cell histones. To date, there are no known histones which contain tryptophan (Bonner et al., 1968), and t)he molecular weight and lysine to valine ratio of components P5-7 (Table 2) are at’ least consistent with the available data on histones (Butler et al., 1968). In addition, Winocour and Robbins (1970) observed an increased synthesis of small, acid-ext’ractable proteins containing lysine but no tryptophan (“histones”) in cells infected by polyoma or SV40 virus. It may be that Winocour and Robbins observed the same components in polyoma-infected cells that we have detected in purified polyoma virions. Could proteolytic cleavage be responsible for the multiple polyoma virion polypeptides which we have observed? There are two variations of this hypothesis. In the first case, accident.al proteolytic “nicking” of component P2 in assembled virions during cell lysis might generate some or all of the smaller polypeptides. However, t’his is unlikely for two reasons. If such proteolytic cleavage is involved, it must always occur at the samesite and to the same extent in view of the quantitative reproducibility of the seven virion polypeptide components (Table 1). In addition, we observed no quant’itative difference in the virion polypept,ide pattern when virus harvested 48 hr after infection, when few of the cells have lysed, and virus harvested 72-96 hr after infection, when t’he cytopathic effect is complete, were compared by SDS-gel electrophoresis. Alternatively, a regular proteolytic cleavage of a hypothetical large precursor polypeptide could give rise to the seven virion polypeptides without exceeding the coding capacity of t’he viral DNA if portions of t(he different polypeptides overlapped each other. Proteolytic cleavage of a high molecular weight precursor polypeptide, although not apparently into overlapping segments, has been suggested to be an important step in the morphogenesis of poliovirus (Jacobson and Baltimore, 1968).

POLYOX4

VIRIOX

Bot,h variations of the proteolytic cleavage hypot,hesis predict that some of the polyoma virion polypeptide component$s will be found to have peptides in common. This prediction is currently being test’ed by comparing t’he t,rypt,ic peptides of separated polyoma virion polypeptide components. The role played by the minor polypeptide component,s in polyoma, virus infection, transformation, and virus particle assembly remains to be determined. ?tlinor components 1’5-7 have a high 1ysine:valine ratio, are essentially absent from empty shells of polyoma virus, and may therefore be present in virions because they bind t’o viral DNA. This binding could be required to reduce the viral DNA to a compact size for subsequent coating with virus capsid protein. Similar small basic “core” proteins have been detected in many different DNA viruses, for example in adenovirus type 5 (Russel et al., 1968) and in T-even bacteriophage (Stone and Cummings, 1970). An interesting and as yet unanswered question is the fate of components P5-7 after virus infecCon. Do they remain associat’ed with the viral DNA after uncoat’ing processes remove the external virion protein components? Would the binding of such components modify the \vay in which viral DNA is initially transcribed by RX,4 polymerase? A final possible explanat’ion for the multiple minor protein components of polyoma virions is suggest*ed by the recent reports of several enzymatic activities associat’ed with different, mammalian viruses. Examples are the D-VA-dependent R-I;A polymerase (liates and I\Ichuslan, 1967), nucleotide phosphohydrolase (i\lunyon et al., 1968; Gold and Dales, 1968), and deoxyribonuclease (Pogo and Dales, 1969) associated wit,h vaccinia virus, and the endonuclease associated wit#h the penton component of adenovirus type 2 (Burlingham et al., 1970), to mention only a few. Thus, minor protein components I?-7 of polyoma virions may turn out’ t)o be associated with as yet undet,ected enzymatic activit,ies in the virions. ACKSOWLEDCMENTS It. is a pleasure to thank Dr. William T. Murakami for his generous gifts of information and experimental materials throughout work. We would also like to thank

the course of this Virginia Lee and

Xi

PROTISINS

Jrlne Hatley

for much helpful assistance i11 maintenance of t,he ~141 ('\lltul?s. and Marlene Bajak for her excellent electron mcroscopy. This research was aided by grant PF-427 (R. R.) from t,he American Cancer Societ’y and a Nate Fellowship (I?. H.), and supported by (irant. No. CA-07592 from the Sational Cancer In?;l.itllle.

Mrs.

preparation and

R EFTiRRXCES

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AND DULBECCO RUSSEL, W. C., LAYER, W. G., and ~~ANDERSON, P. J. (1968). Internal components of adenovirus. Nature (London) 219, 1127-1130. SHAPIRO, A. L., VIRUELA, E., and MAIZEL, J. V., JR. (1967). Molecular weight estimation of polypeptide chains by electrophoresis on SDS-polyacrylamide gels. Bioch,em. Biophys. Res. Commun. 28, 815-820. STONE, K. R., and CUMMINGS, D. J. (1970). Isolation and characterization of two basic int,ernal proteins from T-even bacteriophages. J. Viral. 6, 44&454. THORNE, H. V., and WARDEN, 1). (1967). Electrophoretic evidence for a single protein component in the capsid of polyoma virus. J. Gen. Viral. 1, 135-137. VOGT, M., and DULBECCO, R. (1962). Studies on cells rendered neoplastic by polyoma virus: the problem of the presence of virus-related materials. Virology 16, 41-51. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 44064412. WINOCOUR, E. (1968). Further studies on the incorporation of cell DNA into polyoma-related particles. Virology 34, 571-582. WINOCOUR, E., and ROBBINS, E. (1970). Histone synthesis in polyoma- and SV40-infected cells. Virology 40, 307-315.