74,
VIROLOGY
(1976)
377-385
Polypeptides
of a Viral DNA-Protein Virus-Infected
ATEEF Dbpartement
de Microbiologic,
A. QURESHI Centre
AND
Complex Cells
PIERRE
June
Polyoma
BOURGAUX’
Hospitalier Universituire, Universitt! Que’bec, Canada JlH 5N4 Accepted
from
de Sherbrooke,
Sherbrooke,
9,1976
A nascent viral DNA-protein complex was isolated from cells productively infected with polyoma virus (Py), by a conventional extraction procedure. This complex was purified by velocity sedimentation through neutral sucrose gradients, followed by ionexchange chromatography. After in vitro labeling with iz51, the protein moiety of the purified complex was analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Four polypeptides were thus detected: viral structural polypeptides VPl, VP2, and VP3, and a nonstructural polypeptide of a molecular weight of approximately 70,000. The latter represented as much as 48% of the lz51 radioactivity associated with all four polypeptides. INTRODUCTION
protein complex extracted from Py-infected cells using Triton X-100 (Green et Several investigators have reported that al., 1971). In agreement with previous renascent supercoiled viral DNA can be isoports (Green et al., 1971; Goldstein et al., lated from either Py- or SV40-infected cells 1973; Shmookler et al., 1974; McMillen and as a DNA-protein complex (Green et al., 1974), we have found this com1971; Green, 1972; Goldstein et al., 1973; Consigli, plex to contain both viral DNA forms I and Hall et al., 1973; Frost and Bourgaux, data) and to sediment at 1973; Shmookler et al., 1974). In both in- II (unpublished 55 S in neutral sucrose solution. We report stances, the complexes are described as here on the partial purification and polycontaining several polypeptides (White and Eason, 1971; Goldstein et al., 1973; peptide composition of this complex. Hall et al., 1973; McMillen and Consigli, MATERIALS AND METHODS 1974). The only published report on the protein composition of such a complex sug Virus and cell cultures. The procedures gests that, in the case of Py, it comprises used for propagation and purification of all of the polypeptides present in the virion the TSP-1 variant (Stanners, 1963) of Py (McMillen and Consigli, 19741. Identificahave been described elsewhere (Bourgaux tion of the proteins present in the viral and Bourgaux-Ramoisy, 1971; Bourgaux et complex is important, since it could give us al., 1971). a clue as to the function of the complex Isolation of radioactively labeled comitself. Conceivably, the latter, already a plex. Twenty-six hours after infection (20 product of viral replication, could either plaque-forming units per cell), secondary serve as template for further replication cultures from whole mouse embryos were and/or late transcription, or alternatively medium changed with DMEM (Grand Isrepresent an intermediate in the assembly land Biological) containing 2% calf serum of progeny virus. and either 5 @i/ml of [3H]thymidine (50.8 We have chosen to study the viral DNACi/mmol, ICN) or 20 pCi/ml of [35Slmethionine (510 Ci/mmol, AmershamlSearle), ’ Author to whom requests for reprints should be addressed. or both. When [35Slmethionine labeling 377 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
378
QURESHI
AND
was desired, medium containing one-tenth of the standard concentration of methionine was used. After 6 hr of pulse-labeling, the monolayer-s were washed three times with cold 0.01 M Tris-HCl buffer, pH 7.5, prior to the extraction of the DNA-protein complex following a procedure modified from that of Green et al. (1971). Briefly, each petri dish (50 mm in diameter) received 180 ~1 of 0.25% Triton X-100 in 0.01 M EDTA, 0.01 M Tris-HCl, pH 7.9. After 10 min at room temperature, 20 ~1 of 2 M NaCl was added. The lysate was then centrifuged at 4” in a Sorvall HG-4 rotor for 10 min at 5000 rpm. The supernatant (Triton extract) containing the DNA-protein complex was collected for further characterization. Purification of complex. The Triton extract was layered on a 15 to 30% (w/w) linear sucrose gradient in 0.01 M EDTA, 0.05 M NaCl, 0.01 M Tris-HCl buffer, pH 7.9, formed on top of a 0.5-ml cushion of 50% (w/w) sucrose, and centrifuged in an International B-60 centrifuge for 4 hr at 40,000 rpm (SB283 rotor). At the end of the centrifugation, fractions were collected from the bottom of the tube and assayed for acid-precipitable radioactivity (Bollum, 1959). The 55 S DNA-containing material (See Fig. 1) recovered from the gradient was concentrated and dialyzed against 0.01 M Tris-HCl buffer, pH 8.6, using an Amicon ultrafilter (Diaflo XM50) prior to ion-exchange chromatography on a QAE-Sephadex A-25 (Pharmacia) column (0.5 x 10 cm). A linear gradient of 0 to 1 M KC1 in 0.01 M Tris-HCl, pH 8.6, was applied to the column. Fractions of the effluent containing acid-precipitable radioactivity were concentrated and dialyzed against 0.01 M sodium phosphate buffer, pH 6.9, using the Amicon ultrafilter (Diaflo XM50). Fixation or disruption of the complex. In some experiments, the material recovered from the sucrose gradient was mixed with an equal volume of cold 12% (v/v) formaldehyde (Fisher, analytical grade) solution in 0.01 M Tris-HCl buffer, pH 8.6, and incubated at 4” overnight, as described by Hancock (1970). The fixed complex was then dialyzed against 0.01 M Tris-HCl
BOURGAUX
13
-12 20s i
-10
Y 9 x E
-8
-6
4-
4 d-' -2
4 Ii-
2-
fraction
number
FIG. 1. Sedimentation properties of Py DNAprotein complex. A Triton extract prepared from virus-infected mouse embryo cells labeled for 6 hr with [3H]thymidine was mixed with 14C-labeled Py DNA form I, used as marker, and layered over a 15 to 30% neutral sucrose gradient. Centrifugation was performed for 4 hr at 40,000 rpm in the SB283 rotor of an International B60 centrifuge. The figure shows the distribution of the acid-precipitable radioactivity through the gradient at the end of the run. Sedimentation is from right to left.
buffer, pH 8.6, to remove excess formaldehyde. In other experiments, the fractions from the sucrose gradient containing the complex were dialyzed at 4” for 2 hr against 100 volumes of 0.2 M Na&03-NaHC03 buffer, pH 10.6, containing 3 x 10e3M dithiothreito1 (Friedmann and David, 1972). After the disrupted complex had been charged on the QAE-Sephadex column, the latter was washed thoroughly with 0.01 M Tris-HCl buffer, pH 8.6, prior to chromatography as described above. Zodination. The protein in the complex was labeled with radioactive iodine using Chloramine T, as already described (Frost and Bourgaux, 1975), except that no carrier protein was added. Polyacrylamide gel electrophoresis. The samples were made to 2% SDS and 2% 2mercaptoethanol, and then boiled for 2
POLYOMA
DNA-PROTEIN
min. Phenol red, used as a tracking dye, and 5% glycerol, were added and electrophoresis was conducted on 8- x 0.5-cm cylindrical 10% SDS-polyacrylamide gels at 60 V for 4 to 5 hr, as described for the SDSphosphate system (Maizel, 1971). Following electrophoresis the gels were frozen at -9O”, and l- to 2-mm-thick slices were cut. Each slice was either dried and counted in a Nuclear Chicago gamma counter, or dissolved in 30% HzO, at 60” for 3 to 4 hr and counted in a Beckman LS 250 spectrometer after addition of PPO-POPOP-toluene containing 30% Triton X-100. RESULTS
Sedimentation Properties Protein Complex
of the DNA-
Twenty-six hours after infection, mouse embryo cells were labeled with 13Hlthymidine and lysed with Triton as described in Materials and Methods. The Triton extract was mixed with [14C]thymidinelabeled Py-marker DNA and centrifuged through a 15 to 30% neutral sucrose gradient. As already observed by others (Green et al., 1971; Green, 1972; Goldstein et al., 1973; Hall et al., 1973; Frost and Bourgaux, 1973; Shmookler et al., 19741, the acid-precipitable material from the extract was found to sediment more rapidly than the 20 S marker, at approximately 55 S (Fig. 1). Over 75% of that material was converted into 20 S sedimenting material following protease digestion, suggesting the complex contained mostly covalently closed cyclic viral DNA (unpublished data). As expected, Triton extracts made from similarly labeled, uninfected cells did not reveal any material sedimenting at 55 S (not shown). Purity of the Complex after Sucrose dient Centrifugation
Gra-
Before attempting any characterization of the protein present in the 55 S complex, it was essential to assess whether adequate purification could be achieved by velocity sedimentation through sucrose gradients. The following experiment was thus performed. Infected cells were labeled with either r3H]thymidine, or both 13Hl-
COMPLEX
379
thymidine and [35S]methionine, from the twenty-sixth to thirty-second hr after infection. Mock-infected cells were labeled at the same time with [35S]methionine only. At the end of the pulse, Triton extracts from the various sets of plates were analyzed either separately or after being mixed, by sedimentation through sucrose gradients (Fig. 2). In the extract from the infected cells, [3H]thymidine-labeled material was found that sedimented at 55 S, as observed before. The 35S pattern indicated the presence of material forming a band sedimenting at approximately 30 S, with a leading shoulder overlapping with the position of the DNA-protein complex, as well as that of a large amount of radioactive material remaining at the top of the gradient (Fig. 2a). The doubly labeled material found near the bottom of the tube represents aggregates, as documented below (see for instance, Fig. 5a and b). The :YS pattern registered for the mock-infected cells also revealed a 30 S band which, in contrast, was not leading into the position that the viral complex would have occupied in such a gradient (Fig. 2b). The centrifugation of the mixed extracts did not indicate cosedimentation of protein from the uninfected cells with 55 S viral complex (Fig. 2~). In concordance with the findings of McMillen and Consigli (19741, our data suggest, first, that the complex includes some Wlabeled material and, second, that the presence of this material in the complex is not the result of aggregation occurring during centrifugation. That the complex is not a mere aggregate of DNA and protein is further suggested by the fact that mixing of purified [3Hlthymidine-labeled DNA with a Triton cell extract (see Materials and Methods) does not generate any :‘H-labeled material sedimenting at 55 S (not shown). On the basis of these findings, we decided to investigate the polypeptide composition of the 55 S peak material recovered from sucrose gradient. This material was therefore labeled with lZ51 as described in Materials and Methods. SDS-polyacrylamide gel electrophoresis of the iodinated protein suggested the presence of polypeptides covering a wide range of molecular
QURESHI
380 3
AND BOURGAUX b
fraction
number
FIG. 2. Sedimentation velocity analysis of Triton extracts from infected and mock-infected cells. Triton extracts were prepared as described in Materials and Methods and subjected to centrifugation as described in Fig. 1. The distributions of radioactivity through the gradients were registered for: (a) an extract from infected cells labeled with both [3H]thymidine and [W]methionine; (b) an extract from [Wmethioninelabeled mock-infected cells; (c) an extract from L3H]thymidine-labeled infected cells added to the extract from 135S1methionine-labeled mock-infected cells analyzed in (b). In all instances, pulse-labeling was continuous for the 6 hr preceding Triton extraction. Sedimentation is from right to left.
weights (Fig. 3). This is a reproducible observation that could not be explained by some artifact produced during iodination or electrophoresis, for the polypeptides from similarly labeled Py, run in a parallel tube (not shown), were found to give rise to well-defined peaks, as already observed (Frost and Bourgaux, 1975). The conflict between our results and those of McMillen and Consigli (19741, which suggested a greater purity of the complex after sucrose gradient fractionation, may be only apparent. While we used lz51 to label the protein of the complex in vitro, those authors analyzed a complex labeled in uivo after relatively short pulses of [35Slmethionine. Our procedure would be expected to render radioactive virtually all proteins present at the time of iodination, regardless of the origin or time of synthesis of the respective proteins. Also, we isolated the complex from whole cells rather than from isolated nuclei. From these observations we concluded that sedimentation through sucrose gradients, although effective in removing
most adventitious proteins, was unlikely to yield preparations of the viral complex that would allow determination of its polypeptide composition. Ion-Exchange
Chromatography
In reconstruction experiments, we observed that the DNA-protein complex could be separated from added proteins, including histone, by chromatography on QAE-Sephadex, a strongly basic ion-exchanger. Therefore 55 S material, labeled with [3H]thymidine and [35Slmethionine, was recovered from sucrose gradients, dialyzed, concentrated, and subjected to QAE-Sephadex chromatography (see Materials and Methods). During this fractionation, over 75% of the 3H counts and about 20% of 35S counts recovered from the column as acid-precipitable material were consistently eluted at approximately 0.75 M KC1 (Fig. 4a), suggesting a DNA-protein association. Whereas most of the 35S counts were recovered either in the column wash or at low KC1 concentrations, only a minor proportion of the “H radioac-
POLYOMA
DNA-PROTEIN
18 1 I
16.
14.
12. P 0
381
COMPLEX
that DNA is eluted at a slightly higher KC1 concentration than complex (0.85 M instead of 0.75 M). This highly reproducible finding suggests that QAE-Sephadex chromatography does not result in extensive dissociation of the complex. As a second control, some doubly labeled complex
lo; on
H
f
6-
4.
8
a-2-b b
UP
OJ
IO
20
30
fraction
40
50
60
70
number
FIG. 3. Electrophoresis of polypeptides from 55 S sedimenting material. A Triton extract from infected cells was subjected to velocity sedimentation (see Fig. 1). The fractions comprising the viral complex were pooled and the proteins thereof were labeled with lz51, denatured, and subjected to SDSpolyacrylamide gel electrophoresis as described in Materials and Methods. Following electrophoresis, slices from the gels were counted in a Nuclear Chicago gamma counter. In addition to the radioactivity distribution through the gel, the figure shows the position of Remazol blue-stained (Griffith, 19721 ovalbumin (01 and histone (H) which had been added to the radioactive sample. Migration is from left to right. fractlol
tivity was eluted at 0.85 M KC1 where free DNA was expected (see below). As a first control, some doubly labeled complex was disrupted according to the method of Friedmann and David (19721, mixed with purified “‘P-labeled Py DNA form I and subjected to QAE-Sephadex chromatography (Fig. 4b). From the acid-precipitable 35S radioactivity then recovered from the column, one-third was eluted in the column wash and most of the remainder between 0.2 and 0.3 M KCl. No %l counts were detected in the fractions where both “H-labeled acid-precipitable material and “‘P-labeled DNA were eluted, as expected from complete dissociation of the complex. Comparing the chromatographic patterns registered for undissociated and for dissociated complexes (Fig. 4a and b) suggested
number
FIG. 4. QAE-Sephadex chromatography of Py DNA-protein complex. The fractions from a sucrose gradient containing doubly labeled complex (see Fig. 2al were pooled, dialyzed against 0.01 M TrisHCl buffer, pH 8.6, and concentrated by Amicon ultrafiltration (Diaflo XM50). The concentrate was loaded onto a 0.5 x lo-cm QAE-Sephadex A-25 column. The column was washed with 0.01 M Tris-HCl buffer, pH 8.6 (fractions l-101, before a 0 to 1 M linear gradient of KC1 solution in the same buffer was applied (fractions 11 to 90). To ensure complete elution, the column was finally washed with 1 M KC1 (fractions 91 to 100). (al Elution pattern of 13Hlthymidineand [““Slmethionine-labeled unfixed complex. (bl Elution pattern of complex labeled with both [“Hlthymidine and 13Slmethionine disrupted with alkali and mixed with 3’P-labeled purified Py DNA form I immediately before chromatography. (c) Elution pattern of complex labeled with both 13Hlthymidine and [%lmethionine, fixed with formaldehyde prior to chromatography.
382
QURESHI
AND
BOURGAUX
was fixed with formaldehyde, following the method of Hancock (1970), prior to chromatography. Most of the labeled protein and DNA were then eluted at approximately the same KC1 concentration as the unfixed complex (Fig. 4~). The marked reduction in the 35S counts eluting as free protein is, at least in part, attributable to the extensive dialysis required to remove excess formaldehyde before chromatography. That fixation does not result in a detectable change in the chromatographic properties of the complex further suggests that adsorption onto and elution from QAE-Sephadex does not involve a major loss of protein from the complex. It should be noted that the 35S-labeled material eluting in the column wash migrated during polyacrylamide gel electrophoresis as histone (not shown). Sedimentation Properties of Complex lowing Chromatography
fol-
The protein composition of the complex could not be determined unless the 0.75 M KC1 peak recovered from QAE-Sephadex was extensively dialyzed and concentrated. Despite repeated efforts, the 35S activity left over at the end of this procedure was always too low to allow SDSpolyacrylamide gel electrophoresis to be performed. We thus resorted to labeling of the purified complex with lZ51, following a procedure already used to label the polyoma virion itself (Frost and Bourgaux, 1975). In order to separate the complex from free lz51, the in vitro reaction mixture was centrifuged over a 15 to 30% preparative sucrose gradient immediately after iodination. From 60 to 70% of the bound lz51 was thus found to sediment at 50-55 S, i.e., with a sedimentation coefficient closely similar to that of the complex detected in the original cell extract (Fig. 5a and c). As already noticed in the first sucrose gradient fractionation (see Fig. 2a), some of the bound lz51 sedimented faster than the complex. As further suggested by its protein composition (see next section), this fast-sedimenting material too was likely to represent aggregates. Finally, 10 to 15% of the acid-precipitable lz51 counts were recovered in the top fractions of the gradient.
fraction
number
FIG. 5. Sedimentation properties of Py DNAprotein complex iodinated following chromatography. Complex recovered from the QAE-Sephadex column was labeled with lz51 and immediately thereafter centrifuged through a sucrose gradient, as described in Fig. 1. Fractions from this preparative gradient (a) were pooled as indicated on the figure, mixed with 32P-labeled DNA form I, and recentrifuged under the same conditions (b). A crude Triton extract from infected cells labeled with both [3Hlthymidine and [Wlmethionine (see Fig. 2a) was similarly analyzed (c). The figure shows the distribution of radioactivity through the various gradients. Note that most of the lz51 radioactivity remaining at the top of the preparative gradient (panel a) represents unbound iodine. Sedimentation is from right to left.
In order to confirm that the sedimentation coefficient of the complex was essentially unaffected by purification, the material presumed to represent complex was recovered after sucrose gradient fractionation, mixed with 32P-labeled purified Py
POLYOMA
DNA-PROTEIN
383
COMPLEX
DNA form I used as a marker, and recentrifuged under identical conditions. Most of the acid-precipitable lz51 radioactivity was thus found to sediment as a broad band with a peak fraction corresponding to approximately 52 S, in relation to the 20 S viral DNA (Fig. 5b). The markedly trailing lz51 pattern, contrasting with the profile registered for the marker, suggested partial dissociation of the complex and, possibly, some interaction with the marker itself.
1972; Crawford and Gesteland, 1973; Frost and Bourgaux, 1975). The 125I pattern revealed the presence of at least four labeled polypeptides. Besides radioactive material comigrating with viral structural polypeptides VPl, VP2, and VP3, a polypeptide absent from virus and of approximate molecular weight 70,000 was detected. This estimation of molecular weight is based on distance migration through gel, in relation to bovine serum albumin, haemoglobin, and ovalbumin (not shown). As indicated by Fig. 6, it is also consistent with the SDS-Polyacrylamide Gel Electrophoresis positions of VP1 and of its presumptive dimer in the gels (Friedmann, 1974). This Iodine-labeled complex purified from heretofore unrecognized component was free lz51by velocity sedimentation (see Fig. consistently found as the most prominent 5a) and purified [35S]methionine-labeled of the polyoma virus were mixed, denatured, and one, in a number of preparations suggest subjected to SDS-polyacrylamide gel elec- viral complex. Two observations a genuine controphoresis (Fig. 6). The distribution of 35S that it actually represents stituent of the complex rather than some through the gels clearly indicated the prestrivial contaminant. First, it was not deence of the various polypeptides charactertected in fractions from the QAE-Sephaistic of the virus (Frearson and Crawford, dex column which did not contain the complex, and, second, it copurified with the complex during exclusion chromatography on Sephadex G-200 (not shown). In contrast to McMillen and Consigli (1974), we obviously found little labeled material with the electrophoretic mobility of histone (VP4-7) in the purified complex. Finally, analysis of the material recovered in the bottom fractions from the preparative sucrose gradient (see Fig. 5a) yielded an electrophoretic pattern similar to that shown in Fig. 6. FIG. 6. SDS-polyacrylamide gel electrophoresis of polypeptides from purified complex and virus. Remazol Blue-stained (Griffith, 1972) ovalbumin (0) and histones (H), complex labeled with lrsI after QAE-Sephadex chromatography and purified, [35S]methionine-labeled virus were mixed, denatured, and subjected to electrophoresis as described in Materials and Methods. Slices from the gel were counted in a Beckman LS 250 spectrometer. In addition to the distribution of radioactivity through the gel, the figure shows the position of the stained markers. Note the position of the most prominent lz51 peak with respect to that of the major structural polypeptide VP1 (42,000 to 48,000 daltons; Friedmann and Eckhart, 1974) and to that (D) of the possible dimer of VP1 (86,000 daltons; Roblin et al., 1971). This most prominent peak and the VP1 peak comprised, respectively, 48 and 32% of the lr51 counts detected in the gel.
DISCUSSION
Using a Triton extraction procedure modified from that devised by McMillen and Consigli (1974), we found most of the polyoma virus DNA to be isolated from infected mouse embryo cell cultures in the form of a DNA-protein complex sedimenting at 55 S. There are several precedents for such a finding (Green et al., 1971; Goldstein et al., 1973; Shmookler et al., 1974; McMillen and Consigli, 1974). This complex does not represent a mere aggregate between viral DNA and cellular proteins, as indicated by several lines of evidence. For instance, no appreciable radioactivity was found to sediment with the complex after
the
[3H]thymidine-labeIed
infected
384
QURESHI
AND
cell extract had been mixed with an extract from [35S]methionine-labeled uninfected cells. It could have been argued of course that in extracts from the infected cells, viral DNA is already covered with proteins, rendering further binding impossible. Similar experiments were thus performed in which purified viral DNA was used instead of crude viral complex. In this instance, conversion of the 20 S DNA into 55 S material was not observed. The complex we have isolated after velocity sedimentation of the extracts is thus likely to represent a genuine in vivo product of viral replication. Such a complex would be expected to contain virus-coded and, possibly, cellular proteins. The sucrose gradient analyses we performed using extracts from [35Slmethionine-labeled cells also suggest that sufficient purification of the complex from adventitious proteins could not be achieved by velocity sedimentation only. Therefore, this complex was further purified by ionexchange chromatography prior to analysis of its protein content. Since QAE-Sephadex is a strong anion exchanger, free basic proteins were expected to be excluded from the column, while free acidic proteins and the complex itself would be retained. Two observations suggest that this chromatographic procedure preserved the integrity of DNA-protein complex. First, DNA-containing material was reproducibly eluted from the column at the same salt concentration of eluent, irrespective of whether the complex had been fixed with formaldehyde or not. Second, the sedimentation properties of this material were essentially unchanged after chromatography (Fig. 5). We thus feel justified in considering that QAE-Sephadex chromatography purifies the complex, but does not extensively modify its composition. This last point raises a serious question. In contrast to McMillen and Consigli (19741, we found relatively little histonelike material in the purified complex. In our experiments, some of this was excluded from the column at the start of chromatography, even when the complex had been fixed with formaldehyde. Whether the basic polypeptides eliminated, at this stage of the purification repre-
BOURGAUX
sented either contaminants or genuine constituents of the complex, already detached, would be very difficult to answer unequivocally. Current observations (e.g., Germond et al., 1975) on chromatinlike structures from papovaviruses make it tempting to conclude that histone might be included in the in vivo complex. In this instance, however, we feel it would be prejudicial to conclude a priori that histone was lost from the complex during purification, rather than that the complex was purified from adventitious histone, because histone should be, in any event, a major source of contamination. This appears inevitable in view of its high rate of synthesis following Py (Seehafer and Weil, 1974) or SV40 (Anderson and Gesteland, 1972) infection, its accumulation in the nucleus where the viral DNA replicates and also accumulates (Frost and Bourgaux, 19731, and the expected tendency of such a basic protein to adhere to the negatively charged complex. The presence of a 70,000-dalton protein in purified preparations of the complex is intriguing. No such polypeptide has been reproducibly detected in significant amounts in either virions or DNA-protein complexes from SV40 or polyoma virus. Some preliminary data we have obtained (Gibson and Qureshi, unpublished results) by tryptic mapping clearly indicate that this polypeptide is distinct from both bovine serum albumin, a likely contaminant of cell cultures, and from the major viral structural polypeptide (VPl). Two further possibilities of interest as to the nature of this polypeptide are the untwisting activity (Champoux and Dulbecco, 1972) already detected in the SV40 DNA-protein complex (Sen and Levine, 19741, and T antigen, which both seem to have molecular weights in the range of 70,000 to 100,000 (Del Villano and Defendi, 1973; Tegtmeyer, 1974; Keller and Wendel, 1974). We are presently investigating whether the unidentified component we detected in the complex would exhibit either of these activities. ACKNOWLEDGMENTS lin,
We wish Harvard
to express Medical
our gratitude to Richard School, for the generous
Robhelp
POLYOMA
DNA-PROTEIN
and advice provided at an early stage of this work, and to Edward Bradley for critical review of the manuscript. We also thank Karen Trollope for excellent technical assistance. One of us (A.A.Q.) was a Research Fellow of the Cancer Research Society, Inc., Montreal. This investigation was supported by the Medical Research Council of Canada. REFERENCES C. W., and GESTELAND, R. F. (1972). Pattern of protein synthesis in monkey cells infected by simian virus 40. J. Viral. 9, 758-765. BOLLUM, F. J. (1959). Thermal conversion of nonpriming deoxynucleic acid to primer. J. Biol. Chem. 234, 2733-2734. BOURGAUX, P., and BOURGAUX-RAMOISY, D. (1971). A symmetrical model for polyoma virus DNA replication. J. Mol. Biol. 62, 513-524. BOURGAUX, P., BOURGAUX-RAMOISY, D., and SEILER, P. (1971). The replication of the ringshaped DNA of polyoma virus. II. Identification of molecules at various stages of replication, J. Mol. Biol. 59, 195205. CHAMPOUX, J. J., and DULBECCO, R. (1972). An activity from mammalian cells that untwists superhelical DNA. A possible swivel for DNA replication Proc. Nat. Acad. Sci. USA 69, 143-146. CRAWFORD, L. V., and GESTELAND, R. F. (1973). Synthesis of polyoma proteins in vitro. J. Mol. Biol. 74, 627-634. DEL VILLANO, B. D., and DEFENDI, V. (1973). Characterization of SV40 T antigen. Virology 51,34-46. FREARSON, P., and CRAWFORD, L. V. (1972). Polyoma virus basic proteins. J. Gen. Virol. 14,141-155. FRIEDMANN, T. (1974). Genetic economy of polyoma virus: Capsid proteins are cleavage products of same viral genes. Proc. Nat. Acad. Sci. USA 71, 257-259. FRIEDMANN, T., and DAVID, D. (1972). Structural roles of polyoma virus proteins. J. Virol. 10, 776782. FRIEDMANN, T., and ECKHART, W. (1974). Virion proteins of polyoma temperature-sensitive mutants: Late mutants. Cold Spring Harbor Symp. Quant. Biol. 39, 243-246. FROST, E., and BOURGAUX, P. (1973). Attempts to find a specific intranuclear location for replicating polyoma virus DNA. Canad. J. Biochem. 51,12251228. FROST, E., and BOURGAUX, P. (1975). Decapsidation of polyoma virus. Identification of subviral species. Virology 68, 245-255. GERMOND, J. E., HIRT, B., OUDET, P., GROSS-BELLARDI, M., and CHAMBON, P. (1975). Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc. Nat. Acad. Sci. USA 72, 1843-1847. GOLDSTEIN, D. A., HALL, M. R., and MEINKE, W.
ANDERSON,
COMPLEX
385
(1973). Properties of nucleoprotein complexes containing replicating polyoma DNA. J. Virol. 12, 887-900. GREEN, M. H. (1972). Biosynthetic properties of a polyoma nucleoprotein complex: Evidence for replication sites. J. Viral. 10, 32-41. GREEN, M. H., MILLER, H. I., and HENDLER, S. (1971). Isolation of a polyoma nucleoprotein complex from infected mouse-cell cultures. Proc. Nat. Acad. Sci. USA 68, 1032-1036. GRIFFITH, I. P. (1972). Immediate visualization of proteins in dodecyl sulfate-polyacrylamide gels by prestaining with Remazol dyes. Ann. Biochem. 46, 402-412. HALL, M. R., MEINKE, W., and GOLDSTEIN, D. A. (1973). Nucleoprotein complexes containing replicating simian virus 40 DNA: Comparison with polyoma nucleoprotein complexes. J. Virol. 12, 901-908. HANCOCK, R. (1970). Separation by equilibrium centrifugation in CsCl gradients of density labeled and normal deoxyproteins from chromatin. J. Mol. Biol. 48, 357-360. KELLER, W., and WENDEL, I. (1974). Step wise relaxation of supercoiled SV40 DNA. Cold Spring Harbor Symp. Quant. Biol. 39, 199-208. MAIZEL, J. V., JR. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.1, Vol. 5, pp. 179-246. Academic Press, New York. MCMILLEN, J., and CONSIGLI, R. A. (1974). Characterization of polyoma DNA-protein complex. I. Electrophoretic identification of the proteins in a nucleoprotein complex isolated from polyoma infected cells. J. Virol. 14, 1326-1336. ROBLIN, R., HKRLE, E., and DULBECCO, R. (19711. Polyoma virus proteins. I. Multiple virus components. Virology 45, 555-566. SEEHAFER, J. G., and WEIL, R. (1974). Synthesis of polyoma virus structural polypeptides in mouse kidney cells. Virology 58, 75-85. SEN, A., and LEVINE, A. J. (1974). SV40 nucleoprotein complex activity unwinds superhelical turns in SV40 DNA. Nature (London) 249, 343-344. SHMOOKLER, R. J., Buss, J., and GREEN, M. H. (19741. Properties of the polyoma virus transcription complex obtained from mouse nuclei. Virology 57, 122-127. STANNERS, C. P. (19631. Studies on the transformation of hamster embryo cells in culture by polyoma virus. I. Properties of transformed and normal cells. Virology 21, 448-463. TEGTMEYER, P. (19741. Altered patterns of protein synthesis in infection by SV40 mutants. Cold Spring Harbor Symp. Quant. Biol. 39, 9-15. WHTE, M., and EASON, R. (1971). Nucleoprotein complexes in Simian virus 40-infected cells. J. Virol. 8, 363-371.
VIROLOGY
(1976)
377-385
Polypeptides
of a Viral DNA-Protein Virus-Infected
ATEEF Dbpartement
de Microbiologic,
A. QURESHI Centre
AND
Complex Cells
PIERRE
June
Polyoma
BOURGAUX’
Hospitalier Universituire, Universitt! Que’bec, Canada JlH 5N4 Accepted
from
de Sherbrooke,
Sherbrooke,
9,1976
A nascent viral DNA-protein complex was isolated from cells productively infected with polyoma virus (Py), by a conventional extraction procedure. This complex was purified by velocity sedimentation through neutral sucrose gradients, followed by ionexchange chromatography. After in vitro labeling with iz51, the protein moiety of the purified complex was analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Four polypeptides were thus detected: viral structural polypeptides VPl, VP2, and VP3, and a nonstructural polypeptide of a molecular weight of approximately 70,000. The latter represented as much as 48% of the lz51 radioactivity associated with all four polypeptides. INTRODUCTION
protein complex extracted from Py-infected cells using Triton X-100 (Green et Several investigators have reported that al., 1971). In agreement with previous renascent supercoiled viral DNA can be isoports (Green et al., 1971; Goldstein et al., lated from either Py- or SV40-infected cells 1973; Shmookler et al., 1974; McMillen and as a DNA-protein complex (Green et al., 1974), we have found this com1971; Green, 1972; Goldstein et al., 1973; Consigli, plex to contain both viral DNA forms I and Hall et al., 1973; Frost and Bourgaux, data) and to sediment at 1973; Shmookler et al., 1974). In both in- II (unpublished 55 S in neutral sucrose solution. We report stances, the complexes are described as here on the partial purification and polycontaining several polypeptides (White and Eason, 1971; Goldstein et al., 1973; peptide composition of this complex. Hall et al., 1973; McMillen and Consigli, MATERIALS AND METHODS 1974). The only published report on the protein composition of such a complex sug Virus and cell cultures. The procedures gests that, in the case of Py, it comprises used for propagation and purification of all of the polypeptides present in the virion the TSP-1 variant (Stanners, 1963) of Py (McMillen and Consigli, 19741. Identificahave been described elsewhere (Bourgaux tion of the proteins present in the viral and Bourgaux-Ramoisy, 1971; Bourgaux et complex is important, since it could give us al., 1971). a clue as to the function of the complex Isolation of radioactively labeled comitself. Conceivably, the latter, already a plex. Twenty-six hours after infection (20 product of viral replication, could either plaque-forming units per cell), secondary serve as template for further replication cultures from whole mouse embryos were and/or late transcription, or alternatively medium changed with DMEM (Grand Isrepresent an intermediate in the assembly land Biological) containing 2% calf serum of progeny virus. and either 5 @i/ml of [3H]thymidine (50.8 We have chosen to study the viral DNACi/mmol, ICN) or 20 pCi/ml of [35Slmethionine (510 Ci/mmol, AmershamlSearle), ’ Author to whom requests for reprints should be addressed. or both. When [35Slmethionine labeling 377 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
378
QURESHI
AND
was desired, medium containing one-tenth of the standard concentration of methionine was used. After 6 hr of pulse-labeling, the monolayer-s were washed three times with cold 0.01 M Tris-HCl buffer, pH 7.5, prior to the extraction of the DNA-protein complex following a procedure modified from that of Green et al. (1971). Briefly, each petri dish (50 mm in diameter) received 180 ~1 of 0.25% Triton X-100 in 0.01 M EDTA, 0.01 M Tris-HCl, pH 7.9. After 10 min at room temperature, 20 ~1 of 2 M NaCl was added. The lysate was then centrifuged at 4” in a Sorvall HG-4 rotor for 10 min at 5000 rpm. The supernatant (Triton extract) containing the DNA-protein complex was collected for further characterization. Purification of complex. The Triton extract was layered on a 15 to 30% (w/w) linear sucrose gradient in 0.01 M EDTA, 0.05 M NaCl, 0.01 M Tris-HCl buffer, pH 7.9, formed on top of a 0.5-ml cushion of 50% (w/w) sucrose, and centrifuged in an International B-60 centrifuge for 4 hr at 40,000 rpm (SB283 rotor). At the end of the centrifugation, fractions were collected from the bottom of the tube and assayed for acid-precipitable radioactivity (Bollum, 1959). The 55 S DNA-containing material (See Fig. 1) recovered from the gradient was concentrated and dialyzed against 0.01 M Tris-HCl buffer, pH 8.6, using an Amicon ultrafilter (Diaflo XM50) prior to ion-exchange chromatography on a QAE-Sephadex A-25 (Pharmacia) column (0.5 x 10 cm). A linear gradient of 0 to 1 M KC1 in 0.01 M Tris-HCl, pH 8.6, was applied to the column. Fractions of the effluent containing acid-precipitable radioactivity were concentrated and dialyzed against 0.01 M sodium phosphate buffer, pH 6.9, using the Amicon ultrafilter (Diaflo XM50). Fixation or disruption of the complex. In some experiments, the material recovered from the sucrose gradient was mixed with an equal volume of cold 12% (v/v) formaldehyde (Fisher, analytical grade) solution in 0.01 M Tris-HCl buffer, pH 8.6, and incubated at 4” overnight, as described by Hancock (1970). The fixed complex was then dialyzed against 0.01 M Tris-HCl
BOURGAUX
13
-12 20s i
-10
Y 9 x E
-8
-6
4-
4 d-' -2
4 Ii-
2-
fraction
number
FIG. 1. Sedimentation properties of Py DNAprotein complex. A Triton extract prepared from virus-infected mouse embryo cells labeled for 6 hr with [3H]thymidine was mixed with 14C-labeled Py DNA form I, used as marker, and layered over a 15 to 30% neutral sucrose gradient. Centrifugation was performed for 4 hr at 40,000 rpm in the SB283 rotor of an International B60 centrifuge. The figure shows the distribution of the acid-precipitable radioactivity through the gradient at the end of the run. Sedimentation is from right to left.
buffer, pH 8.6, to remove excess formaldehyde. In other experiments, the fractions from the sucrose gradient containing the complex were dialyzed at 4” for 2 hr against 100 volumes of 0.2 M Na&03-NaHC03 buffer, pH 10.6, containing 3 x 10e3M dithiothreito1 (Friedmann and David, 1972). After the disrupted complex had been charged on the QAE-Sephadex column, the latter was washed thoroughly with 0.01 M Tris-HCl buffer, pH 8.6, prior to chromatography as described above. Zodination. The protein in the complex was labeled with radioactive iodine using Chloramine T, as already described (Frost and Bourgaux, 1975), except that no carrier protein was added. Polyacrylamide gel electrophoresis. The samples were made to 2% SDS and 2% 2mercaptoethanol, and then boiled for 2
POLYOMA
DNA-PROTEIN
min. Phenol red, used as a tracking dye, and 5% glycerol, were added and electrophoresis was conducted on 8- x 0.5-cm cylindrical 10% SDS-polyacrylamide gels at 60 V for 4 to 5 hr, as described for the SDSphosphate system (Maizel, 1971). Following electrophoresis the gels were frozen at -9O”, and l- to 2-mm-thick slices were cut. Each slice was either dried and counted in a Nuclear Chicago gamma counter, or dissolved in 30% HzO, at 60” for 3 to 4 hr and counted in a Beckman LS 250 spectrometer after addition of PPO-POPOP-toluene containing 30% Triton X-100. RESULTS
Sedimentation Properties Protein Complex
of the DNA-
Twenty-six hours after infection, mouse embryo cells were labeled with 13Hlthymidine and lysed with Triton as described in Materials and Methods. The Triton extract was mixed with [14C]thymidinelabeled Py-marker DNA and centrifuged through a 15 to 30% neutral sucrose gradient. As already observed by others (Green et al., 1971; Green, 1972; Goldstein et al., 1973; Hall et al., 1973; Frost and Bourgaux, 1973; Shmookler et al., 19741, the acid-precipitable material from the extract was found to sediment more rapidly than the 20 S marker, at approximately 55 S (Fig. 1). Over 75% of that material was converted into 20 S sedimenting material following protease digestion, suggesting the complex contained mostly covalently closed cyclic viral DNA (unpublished data). As expected, Triton extracts made from similarly labeled, uninfected cells did not reveal any material sedimenting at 55 S (not shown). Purity of the Complex after Sucrose dient Centrifugation
Gra-
Before attempting any characterization of the protein present in the 55 S complex, it was essential to assess whether adequate purification could be achieved by velocity sedimentation through sucrose gradients. The following experiment was thus performed. Infected cells were labeled with either r3H]thymidine, or both 13Hl-
COMPLEX
379
thymidine and [35S]methionine, from the twenty-sixth to thirty-second hr after infection. Mock-infected cells were labeled at the same time with [35S]methionine only. At the end of the pulse, Triton extracts from the various sets of plates were analyzed either separately or after being mixed, by sedimentation through sucrose gradients (Fig. 2). In the extract from the infected cells, [3H]thymidine-labeled material was found that sedimented at 55 S, as observed before. The 35S pattern indicated the presence of material forming a band sedimenting at approximately 30 S, with a leading shoulder overlapping with the position of the DNA-protein complex, as well as that of a large amount of radioactive material remaining at the top of the gradient (Fig. 2a). The doubly labeled material found near the bottom of the tube represents aggregates, as documented below (see for instance, Fig. 5a and b). The :YS pattern registered for the mock-infected cells also revealed a 30 S band which, in contrast, was not leading into the position that the viral complex would have occupied in such a gradient (Fig. 2b). The centrifugation of the mixed extracts did not indicate cosedimentation of protein from the uninfected cells with 55 S viral complex (Fig. 2~). In concordance with the findings of McMillen and Consigli (19741, our data suggest, first, that the complex includes some Wlabeled material and, second, that the presence of this material in the complex is not the result of aggregation occurring during centrifugation. That the complex is not a mere aggregate of DNA and protein is further suggested by the fact that mixing of purified [3Hlthymidine-labeled DNA with a Triton cell extract (see Materials and Methods) does not generate any :‘H-labeled material sedimenting at 55 S (not shown). On the basis of these findings, we decided to investigate the polypeptide composition of the 55 S peak material recovered from sucrose gradient. This material was therefore labeled with lZ51 as described in Materials and Methods. SDS-polyacrylamide gel electrophoresis of the iodinated protein suggested the presence of polypeptides covering a wide range of molecular
QURESHI
380 3
AND BOURGAUX b
fraction
number
FIG. 2. Sedimentation velocity analysis of Triton extracts from infected and mock-infected cells. Triton extracts were prepared as described in Materials and Methods and subjected to centrifugation as described in Fig. 1. The distributions of radioactivity through the gradients were registered for: (a) an extract from infected cells labeled with both [3H]thymidine and [W]methionine; (b) an extract from [Wmethioninelabeled mock-infected cells; (c) an extract from L3H]thymidine-labeled infected cells added to the extract from 135S1methionine-labeled mock-infected cells analyzed in (b). In all instances, pulse-labeling was continuous for the 6 hr preceding Triton extraction. Sedimentation is from right to left.
weights (Fig. 3). This is a reproducible observation that could not be explained by some artifact produced during iodination or electrophoresis, for the polypeptides from similarly labeled Py, run in a parallel tube (not shown), were found to give rise to well-defined peaks, as already observed (Frost and Bourgaux, 1975). The conflict between our results and those of McMillen and Consigli (19741, which suggested a greater purity of the complex after sucrose gradient fractionation, may be only apparent. While we used lz51 to label the protein of the complex in vitro, those authors analyzed a complex labeled in uivo after relatively short pulses of [35Slmethionine. Our procedure would be expected to render radioactive virtually all proteins present at the time of iodination, regardless of the origin or time of synthesis of the respective proteins. Also, we isolated the complex from whole cells rather than from isolated nuclei. From these observations we concluded that sedimentation through sucrose gradients, although effective in removing
most adventitious proteins, was unlikely to yield preparations of the viral complex that would allow determination of its polypeptide composition. Ion-Exchange
Chromatography
In reconstruction experiments, we observed that the DNA-protein complex could be separated from added proteins, including histone, by chromatography on QAE-Sephadex, a strongly basic ion-exchanger. Therefore 55 S material, labeled with [3H]thymidine and [35Slmethionine, was recovered from sucrose gradients, dialyzed, concentrated, and subjected to QAE-Sephadex chromatography (see Materials and Methods). During this fractionation, over 75% of the 3H counts and about 20% of 35S counts recovered from the column as acid-precipitable material were consistently eluted at approximately 0.75 M KC1 (Fig. 4a), suggesting a DNA-protein association. Whereas most of the 35S counts were recovered either in the column wash or at low KC1 concentrations, only a minor proportion of the “H radioac-
POLYOMA
DNA-PROTEIN
18 1 I
16.
14.
12. P 0
381
COMPLEX
that DNA is eluted at a slightly higher KC1 concentration than complex (0.85 M instead of 0.75 M). This highly reproducible finding suggests that QAE-Sephadex chromatography does not result in extensive dissociation of the complex. As a second control, some doubly labeled complex
lo; on
H
f
6-
4.
8
a-2-b b
UP
OJ
IO
20
30
fraction
40
50
60
70
number
FIG. 3. Electrophoresis of polypeptides from 55 S sedimenting material. A Triton extract from infected cells was subjected to velocity sedimentation (see Fig. 1). The fractions comprising the viral complex were pooled and the proteins thereof were labeled with lz51, denatured, and subjected to SDSpolyacrylamide gel electrophoresis as described in Materials and Methods. Following electrophoresis, slices from the gels were counted in a Nuclear Chicago gamma counter. In addition to the radioactivity distribution through the gel, the figure shows the position of Remazol blue-stained (Griffith, 19721 ovalbumin (01 and histone (H) which had been added to the radioactive sample. Migration is from left to right. fractlol
tivity was eluted at 0.85 M KC1 where free DNA was expected (see below). As a first control, some doubly labeled complex was disrupted according to the method of Friedmann and David (19721, mixed with purified “‘P-labeled Py DNA form I and subjected to QAE-Sephadex chromatography (Fig. 4b). From the acid-precipitable 35S radioactivity then recovered from the column, one-third was eluted in the column wash and most of the remainder between 0.2 and 0.3 M KCl. No %l counts were detected in the fractions where both “H-labeled acid-precipitable material and “‘P-labeled DNA were eluted, as expected from complete dissociation of the complex. Comparing the chromatographic patterns registered for undissociated and for dissociated complexes (Fig. 4a and b) suggested
number
FIG. 4. QAE-Sephadex chromatography of Py DNA-protein complex. The fractions from a sucrose gradient containing doubly labeled complex (see Fig. 2al were pooled, dialyzed against 0.01 M TrisHCl buffer, pH 8.6, and concentrated by Amicon ultrafiltration (Diaflo XM50). The concentrate was loaded onto a 0.5 x lo-cm QAE-Sephadex A-25 column. The column was washed with 0.01 M Tris-HCl buffer, pH 8.6 (fractions l-101, before a 0 to 1 M linear gradient of KC1 solution in the same buffer was applied (fractions 11 to 90). To ensure complete elution, the column was finally washed with 1 M KC1 (fractions 91 to 100). (al Elution pattern of 13Hlthymidineand [““Slmethionine-labeled unfixed complex. (bl Elution pattern of complex labeled with both [“Hlthymidine and 13Slmethionine disrupted with alkali and mixed with 3’P-labeled purified Py DNA form I immediately before chromatography. (c) Elution pattern of complex labeled with both 13Hlthymidine and [%lmethionine, fixed with formaldehyde prior to chromatography.
382
QURESHI
AND
BOURGAUX
was fixed with formaldehyde, following the method of Hancock (1970), prior to chromatography. Most of the labeled protein and DNA were then eluted at approximately the same KC1 concentration as the unfixed complex (Fig. 4~). The marked reduction in the 35S counts eluting as free protein is, at least in part, attributable to the extensive dialysis required to remove excess formaldehyde before chromatography. That fixation does not result in a detectable change in the chromatographic properties of the complex further suggests that adsorption onto and elution from QAE-Sephadex does not involve a major loss of protein from the complex. It should be noted that the 35S-labeled material eluting in the column wash migrated during polyacrylamide gel electrophoresis as histone (not shown). Sedimentation Properties of Complex lowing Chromatography
fol-
The protein composition of the complex could not be determined unless the 0.75 M KC1 peak recovered from QAE-Sephadex was extensively dialyzed and concentrated. Despite repeated efforts, the 35S activity left over at the end of this procedure was always too low to allow SDSpolyacrylamide gel electrophoresis to be performed. We thus resorted to labeling of the purified complex with lZ51, following a procedure already used to label the polyoma virion itself (Frost and Bourgaux, 1975). In order to separate the complex from free lz51, the in vitro reaction mixture was centrifuged over a 15 to 30% preparative sucrose gradient immediately after iodination. From 60 to 70% of the bound lz51 was thus found to sediment at 50-55 S, i.e., with a sedimentation coefficient closely similar to that of the complex detected in the original cell extract (Fig. 5a and c). As already noticed in the first sucrose gradient fractionation (see Fig. 2a), some of the bound lz51 sedimented faster than the complex. As further suggested by its protein composition (see next section), this fast-sedimenting material too was likely to represent aggregates. Finally, 10 to 15% of the acid-precipitable lz51 counts were recovered in the top fractions of the gradient.
fraction
number
FIG. 5. Sedimentation properties of Py DNAprotein complex iodinated following chromatography. Complex recovered from the QAE-Sephadex column was labeled with lz51 and immediately thereafter centrifuged through a sucrose gradient, as described in Fig. 1. Fractions from this preparative gradient (a) were pooled as indicated on the figure, mixed with 32P-labeled DNA form I, and recentrifuged under the same conditions (b). A crude Triton extract from infected cells labeled with both [3Hlthymidine and [Wlmethionine (see Fig. 2a) was similarly analyzed (c). The figure shows the distribution of radioactivity through the various gradients. Note that most of the lz51 radioactivity remaining at the top of the preparative gradient (panel a) represents unbound iodine. Sedimentation is from right to left.
In order to confirm that the sedimentation coefficient of the complex was essentially unaffected by purification, the material presumed to represent complex was recovered after sucrose gradient fractionation, mixed with 32P-labeled purified Py
POLYOMA
DNA-PROTEIN
383
COMPLEX
DNA form I used as a marker, and recentrifuged under identical conditions. Most of the acid-precipitable lz51 radioactivity was thus found to sediment as a broad band with a peak fraction corresponding to approximately 52 S, in relation to the 20 S viral DNA (Fig. 5b). The markedly trailing lz51 pattern, contrasting with the profile registered for the marker, suggested partial dissociation of the complex and, possibly, some interaction with the marker itself.
1972; Crawford and Gesteland, 1973; Frost and Bourgaux, 1975). The 125I pattern revealed the presence of at least four labeled polypeptides. Besides radioactive material comigrating with viral structural polypeptides VPl, VP2, and VP3, a polypeptide absent from virus and of approximate molecular weight 70,000 was detected. This estimation of molecular weight is based on distance migration through gel, in relation to bovine serum albumin, haemoglobin, and ovalbumin (not shown). As indicated by Fig. 6, it is also consistent with the SDS-Polyacrylamide Gel Electrophoresis positions of VP1 and of its presumptive dimer in the gels (Friedmann, 1974). This Iodine-labeled complex purified from heretofore unrecognized component was free lz51by velocity sedimentation (see Fig. consistently found as the most prominent 5a) and purified [35S]methionine-labeled of the polyoma virus were mixed, denatured, and one, in a number of preparations suggest subjected to SDS-polyacrylamide gel elec- viral complex. Two observations a genuine controphoresis (Fig. 6). The distribution of 35S that it actually represents stituent of the complex rather than some through the gels clearly indicated the prestrivial contaminant. First, it was not deence of the various polypeptides charactertected in fractions from the QAE-Sephaistic of the virus (Frearson and Crawford, dex column which did not contain the complex, and, second, it copurified with the complex during exclusion chromatography on Sephadex G-200 (not shown). In contrast to McMillen and Consigli (1974), we obviously found little labeled material with the electrophoretic mobility of histone (VP4-7) in the purified complex. Finally, analysis of the material recovered in the bottom fractions from the preparative sucrose gradient (see Fig. 5a) yielded an electrophoretic pattern similar to that shown in Fig. 6. FIG. 6. SDS-polyacrylamide gel electrophoresis of polypeptides from purified complex and virus. Remazol Blue-stained (Griffith, 1972) ovalbumin (0) and histones (H), complex labeled with lrsI after QAE-Sephadex chromatography and purified, [35S]methionine-labeled virus were mixed, denatured, and subjected to electrophoresis as described in Materials and Methods. Slices from the gel were counted in a Beckman LS 250 spectrometer. In addition to the distribution of radioactivity through the gel, the figure shows the position of the stained markers. Note the position of the most prominent lz51 peak with respect to that of the major structural polypeptide VP1 (42,000 to 48,000 daltons; Friedmann and Eckhart, 1974) and to that (D) of the possible dimer of VP1 (86,000 daltons; Roblin et al., 1971). This most prominent peak and the VP1 peak comprised, respectively, 48 and 32% of the lr51 counts detected in the gel.
DISCUSSION
Using a Triton extraction procedure modified from that devised by McMillen and Consigli (1974), we found most of the polyoma virus DNA to be isolated from infected mouse embryo cell cultures in the form of a DNA-protein complex sedimenting at 55 S. There are several precedents for such a finding (Green et al., 1971; Goldstein et al., 1973; Shmookler et al., 1974; McMillen and Consigli, 1974). This complex does not represent a mere aggregate between viral DNA and cellular proteins, as indicated by several lines of evidence. For instance, no appreciable radioactivity was found to sediment with the complex after
the
[3H]thymidine-labeIed
infected
384
QURESHI
AND
cell extract had been mixed with an extract from [35S]methionine-labeled uninfected cells. It could have been argued of course that in extracts from the infected cells, viral DNA is already covered with proteins, rendering further binding impossible. Similar experiments were thus performed in which purified viral DNA was used instead of crude viral complex. In this instance, conversion of the 20 S DNA into 55 S material was not observed. The complex we have isolated after velocity sedimentation of the extracts is thus likely to represent a genuine in vivo product of viral replication. Such a complex would be expected to contain virus-coded and, possibly, cellular proteins. The sucrose gradient analyses we performed using extracts from [35Slmethionine-labeled cells also suggest that sufficient purification of the complex from adventitious proteins could not be achieved by velocity sedimentation only. Therefore, this complex was further purified by ionexchange chromatography prior to analysis of its protein content. Since QAE-Sephadex is a strong anion exchanger, free basic proteins were expected to be excluded from the column, while free acidic proteins and the complex itself would be retained. Two observations suggest that this chromatographic procedure preserved the integrity of DNA-protein complex. First, DNA-containing material was reproducibly eluted from the column at the same salt concentration of eluent, irrespective of whether the complex had been fixed with formaldehyde or not. Second, the sedimentation properties of this material were essentially unchanged after chromatography (Fig. 5). We thus feel justified in considering that QAE-Sephadex chromatography purifies the complex, but does not extensively modify its composition. This last point raises a serious question. In contrast to McMillen and Consigli (19741, we found relatively little histonelike material in the purified complex. In our experiments, some of this was excluded from the column at the start of chromatography, even when the complex had been fixed with formaldehyde. Whether the basic polypeptides eliminated, at this stage of the purification repre-
BOURGAUX
sented either contaminants or genuine constituents of the complex, already detached, would be very difficult to answer unequivocally. Current observations (e.g., Germond et al., 1975) on chromatinlike structures from papovaviruses make it tempting to conclude that histone might be included in the in vivo complex. In this instance, however, we feel it would be prejudicial to conclude a priori that histone was lost from the complex during purification, rather than that the complex was purified from adventitious histone, because histone should be, in any event, a major source of contamination. This appears inevitable in view of its high rate of synthesis following Py (Seehafer and Weil, 1974) or SV40 (Anderson and Gesteland, 1972) infection, its accumulation in the nucleus where the viral DNA replicates and also accumulates (Frost and Bourgaux, 19731, and the expected tendency of such a basic protein to adhere to the negatively charged complex. The presence of a 70,000-dalton protein in purified preparations of the complex is intriguing. No such polypeptide has been reproducibly detected in significant amounts in either virions or DNA-protein complexes from SV40 or polyoma virus. Some preliminary data we have obtained (Gibson and Qureshi, unpublished results) by tryptic mapping clearly indicate that this polypeptide is distinct from both bovine serum albumin, a likely contaminant of cell cultures, and from the major viral structural polypeptide (VPl). Two further possibilities of interest as to the nature of this polypeptide are the untwisting activity (Champoux and Dulbecco, 1972) already detected in the SV40 DNA-protein complex (Sen and Levine, 19741, and T antigen, which both seem to have molecular weights in the range of 70,000 to 100,000 (Del Villano and Defendi, 1973; Tegtmeyer, 1974; Keller and Wendel, 1974). We are presently investigating whether the unidentified component we detected in the complex would exhibit either of these activities. ACKNOWLEDGMENTS lin,
We wish Harvard
to express Medical
our gratitude to Richard School, for the generous
Robhelp
POLYOMA
DNA-PROTEIN
and advice provided at an early stage of this work, and to Edward Bradley for critical review of the manuscript. We also thank Karen Trollope for excellent technical assistance. One of us (A.A.Q.) was a Research Fellow of the Cancer Research Society, Inc., Montreal. This investigation was supported by the Medical Research Council of Canada. REFERENCES C. W., and GESTELAND, R. F. (1972). Pattern of protein synthesis in monkey cells infected by simian virus 40. J. Viral. 9, 758-765. BOLLUM, F. J. (1959). Thermal conversion of nonpriming deoxynucleic acid to primer. J. Biol. Chem. 234, 2733-2734. BOURGAUX, P., and BOURGAUX-RAMOISY, D. (1971). A symmetrical model for polyoma virus DNA replication. J. Mol. Biol. 62, 513-524. BOURGAUX, P., BOURGAUX-RAMOISY, D., and SEILER, P. (1971). The replication of the ringshaped DNA of polyoma virus. II. Identification of molecules at various stages of replication, J. Mol. Biol. 59, 195205. CHAMPOUX, J. J., and DULBECCO, R. (1972). An activity from mammalian cells that untwists superhelical DNA. A possible swivel for DNA replication Proc. Nat. Acad. Sci. USA 69, 143-146. CRAWFORD, L. V., and GESTELAND, R. F. (1973). Synthesis of polyoma proteins in vitro. J. Mol. Biol. 74, 627-634. DEL VILLANO, B. D., and DEFENDI, V. (1973). Characterization of SV40 T antigen. Virology 51,34-46. FREARSON, P., and CRAWFORD, L. V. (1972). Polyoma virus basic proteins. J. Gen. Virol. 14,141-155. FRIEDMANN, T. (1974). Genetic economy of polyoma virus: Capsid proteins are cleavage products of same viral genes. Proc. Nat. Acad. Sci. USA 71, 257-259. FRIEDMANN, T., and DAVID, D. (1972). Structural roles of polyoma virus proteins. J. Virol. 10, 776782. FRIEDMANN, T., and ECKHART, W. (1974). Virion proteins of polyoma temperature-sensitive mutants: Late mutants. Cold Spring Harbor Symp. Quant. Biol. 39, 243-246. FROST, E., and BOURGAUX, P. (1973). Attempts to find a specific intranuclear location for replicating polyoma virus DNA. Canad. J. Biochem. 51,12251228. FROST, E., and BOURGAUX, P. (1975). Decapsidation of polyoma virus. Identification of subviral species. Virology 68, 245-255. GERMOND, J. E., HIRT, B., OUDET, P., GROSS-BELLARDI, M., and CHAMBON, P. (1975). Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc. Nat. Acad. Sci. USA 72, 1843-1847. GOLDSTEIN, D. A., HALL, M. R., and MEINKE, W.
ANDERSON,
COMPLEX
385
(1973). Properties of nucleoprotein complexes containing replicating polyoma DNA. J. Virol. 12, 887-900. GREEN, M. H. (1972). Biosynthetic properties of a polyoma nucleoprotein complex: Evidence for replication sites. J. Viral. 10, 32-41. GREEN, M. H., MILLER, H. I., and HENDLER, S. (1971). Isolation of a polyoma nucleoprotein complex from infected mouse-cell cultures. Proc. Nat. Acad. Sci. USA 68, 1032-1036. GRIFFITH, I. P. (1972). Immediate visualization of proteins in dodecyl sulfate-polyacrylamide gels by prestaining with Remazol dyes. Ann. Biochem. 46, 402-412. HALL, M. R., MEINKE, W., and GOLDSTEIN, D. A. (1973). Nucleoprotein complexes containing replicating simian virus 40 DNA: Comparison with polyoma nucleoprotein complexes. J. Virol. 12, 901-908. HANCOCK, R. (1970). Separation by equilibrium centrifugation in CsCl gradients of density labeled and normal deoxyproteins from chromatin. J. Mol. Biol. 48, 357-360. KELLER, W., and WENDEL, I. (1974). Step wise relaxation of supercoiled SV40 DNA. Cold Spring Harbor Symp. Quant. Biol. 39, 199-208. MAIZEL, J. V., JR. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.1, Vol. 5, pp. 179-246. Academic Press, New York. MCMILLEN, J., and CONSIGLI, R. A. (1974). Characterization of polyoma DNA-protein complex. I. Electrophoretic identification of the proteins in a nucleoprotein complex isolated from polyoma infected cells. J. Virol. 14, 1326-1336. ROBLIN, R., HKRLE, E., and DULBECCO, R. (19711. Polyoma virus proteins. I. Multiple virus components. Virology 45, 555-566. SEEHAFER, J. G., and WEIL, R. (1974). Synthesis of polyoma virus structural polypeptides in mouse kidney cells. Virology 58, 75-85. SEN, A., and LEVINE, A. J. (1974). SV40 nucleoprotein complex activity unwinds superhelical turns in SV40 DNA. Nature (London) 249, 343-344. SHMOOKLER, R. J., Buss, J., and GREEN, M. H. (19741. Properties of the polyoma virus transcription complex obtained from mouse nuclei. Virology 57, 122-127. STANNERS, C. P. (19631. Studies on the transformation of hamster embryo cells in culture by polyoma virus. I. Properties of transformed and normal cells. Virology 21, 448-463. TEGTMEYER, P. (19741. Altered patterns of protein synthesis in infection by SV40 mutants. Cold Spring Harbor Symp. Quant. Biol. 39, 9-15. WHTE, M., and EASON, R. (1971). Nucleoprotein complexes in Simian virus 40-infected cells. J. Virol. 8, 363-371.
