JOURNAL OF VIROLOGY, Sept. 1977, p. 692-699 Copyright C 1977 American Society for Microbiology
Vol. 23, No. 3 Printed in U.S.A.
Simian Virus 40-Specific Ribosome-Binding Proteins Induced by a Nondefective Adenovirus 2-Simian Virus 40 Hybrid GILBERT JAY,* FRANCIS T. JAY, ROBERT M. FRIEDMAN, AND ARTHUR S. LEVINE Pediatric Oncology Branch, National Cancer Institute, and Laboratory of Experimental Pathology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received for publication 3 March 1977
We have studied the intracellular distribution of the two simian virus 40specific proteins, with apparent molecular weights of 56,000 and 42,000, detectable in human KB cells infected by a nondefective adenovirus 2-simian virus 40 hybrid, Ad2+ND2. After a 20-min pulse of ['5S]methionine, about two-thirds of the newly synthesized 56K protein and one-third of the 42K protein were found localized on the plasma membrane. The remainder of each protein was found in the cytoplasm, whereas the nuclear fraction was virtually free of either component. A significant portion of both proteins present in the cytoplasmic fraction was completed to the 40S ribosomal subunits and was not removed by treatment with 0.5 M KCl. Moreover, the portion that was found free in the cytoplasm could bind preferentially and quantitatively to purified 40S ribosomes in vitro, leading us to propose that these simian virus 40 proteins may act as translational control elements in cells.
Simian virus 40 (SV40) induces three dis- distribution of the SV40-specific proteins syntinct immunological activities detectable in thesized by one of the nondefective hybrids, either transformed or productively infected Ad2+ND2 (which contains the SV40 DNA segcells: T antigen (3, 27), tumor-specific trans- ment 0.11 to 0.44 map unit from the endonuplantation antigen (8, 12), and U antigen (20). clease R-EcoRI cleavage site), with the hope The relationship between each of these anti- that knowledge about their "functional" localgens and their exact function in cells remain ization may permit the design of cell-free sysspeculative. tems that would elucidate the specific in vivo Study of these early SV40 antigens is techni- function of the early SV40 antigens. cally difficult because SV40 not only fails to MATERIALS AND METHODS inhibit host protein synthesis upon infection, but actually stimulates certain host cell biosynViruses. Ad2, Ad2+ND,, and Ad2+ND2 stocks thetic activities (14). Consequently, the amount were grown in monolayer cultures of human KB of early viral antigens present at any time after cells, infected at a multiplicity of 5 PFU/cell, and infection is quite small compared with the purified by twice banding on CsCl gradients after background of newly synthesized host proteins; extraction from cell sonic extracts with Freon 113 therefore, their purification has not been (25). The stock viruses were plaque titrated on monolayers of primary human embryonic kidney cells achieved. The nondefective adenovirus 2 (Ad2)-SV40 (19). and labeling of cells. Human KB cells hybrid viruses appear to form an ideal system in Infection monolayers were infected with purified Ad2, for study of the function of various SV40 early Ad2+ND,, or Ad2+ND2 at a multiplicity of 20 PFU/ antigens. These hybrid viruses contain differ- cell. Twenty-four hours postinfection, the monolayent amounts of SV40 DNA covalently inserted ers were rinsed and incubated at 370C in Earle at a unique site in the Ad2 genome (21). As a balanced salt solution containing 5% minimum esresult, several of these hybrid viruses have ac- sential medium, 2% fetal calf serum, and 40 ,uCi of Lquired the genetic information needed to code [35S]methionine per ml (300 to 400 Ci/mmol). After 10 to 20 min, the cells were rinsed with phosphatefor the various early SV40-specific immunologi- buffered saline and scraped from the bottles. cal activities (19). In contrast to SV40 infection, Subcellular fractionation. Washed cells were sushybrid virus infection inhibits host protein syn- pended in a buffer 10 mM Tris-hydrothesis, maximizing the opportunity to isolate chloride (pH 7.4), 10containing mM KCl, 2 mM MgCl2, and 2 SV40-specific proteins. mM dithiothreitol, and were homogenized with a We have attempted to study the intracellular glass Dounce tissue grinder until more than 95% of 692
VOL. 23, 1977
SV40-SPECIFIC RIBOSOME-BINDING PROTEINS
the cells were seen to be broken under the phasecontrast microscope (eight strokes). The cell lysate was spun at 800 x g for 15 min, and both the supernatant and the pellet were saved for further fractionation. (i) Plasma membranes. The surface membranes and nuclei in the pellet from the low-speed centrifugation were separated by the dextran-polyethylene glycol two-phase system as described by Brunette and Till (4), except that 10 mM NaCl was present instead of ZnCl2. Upon separation of the two phases by centrifugation at 11,700 x g for 10 min, the membranes were found at the interphase. The nuclear pellet was saved for subsequent fractionation, while the membrane fraction was further purified as described (4). Purified membranes were completely free of nuclei when examined by phase-contrast microscopy. (ii) Nuclei and nuclear wash. The nuclear pellet from the first two-phase separation was again suspended in equal volumes of the two phases by homogenization in a Dounce tissue grinder. Any contaminating surface membrane was separated from the nuclei upon phase separation by centrifugation. The nuclear pellet was suspended in a buffer composed of 10 mM Tris-hydrochloride (pH 6.8), 150 mM KCl, 5 mM MgCl2, and 1% Triton X-100, again by homogenization in a Dounce tissue grinder. After 10 min at 4VC, the washed nuclei were separated from the nuclear wash by centrifugation. Suspended nuclei remained fully intact and free of membranous material as visualized by phase-contrast microscopy.
(iii) Cytoplasmic fractions. The supernatant from the initial low-speed centrifugation of the cell homogenate was further centrifuged for 30 min at 30,000 x g. The resultant supernatant, containing the cytoplasmic fraction, was further fractionated by centrifugation for 4 h at 100,000 x g. The resultant supernatant was saved and the pellet was suspended by stirring in a low-salt buffer (buffer A) containing 10 mM Tris-hydrochloride (pH 7.4), 50 mM KCl, 2 mM MgCl2, and 2 mM dithiothreitol. Ribosomal subunits. The suspended 100,000 x g pellet containing the ribosomes was fractionated on 10 to 40% (wt/wt) sucrose gradients made up in buffer A. About 25 A260 units were layered onto each 12.5-ml gradient, and centrifugation was for 5 h at 40,000 rpm (284,000 x g) in a Spinco SW40 rotor. The gradients were collected with an ISCO gradient analyzer, and a portion from each fraction was analyzed for total trichloroacetic acid-precipitable radioactivity. The fractions corresponding to the 40S region were pooled. Half of the pooled fraction was dialyzed against buffer A and the other half against a highsalt buffer (the same as buffer A but containing 500 mM KCl). Both samples were concentrated by sprinkling Sephadex G-150 over the dialysis bag, and they were further purified on 12.5-ml 10 to 40% (wt/wt) sucrose gradients made up in the corresponding buffer. After centrifugation for 5 h at 40,000 rpm (284,000 x g), fractions were collected, and a portion from each fraction was assayed for radioactivity. Because of the change in conformation and the loss of significant amounts of ribosome-
693
associated protein factors induced by high-salt conditions, the 40S subunits appeared to sediment at a lower s value in the presence of high salt (see Fig. 3b). In vitro ribosome binding. The 100,000 x g supernatant was dialyzed against a buffer containing 50 mM Tris-hydrochloride (pH 7.4), 100 mM KCl, 2 mM MgCl2, and 2 mM dithiothreitol, and it was subsequently incubated at 37°C for 30 min with 1.0 A260 unit of 40S ribosomes, obtained from either mockor Ad2-infected cells and purified on sucrose gradients. The reaction products were analyzed on 10 to 40% (wt/wt) sucrose gradients made up in buffer A. Polyacrylamide gel electrophoresis. All samples for analysis contained 62 mM Tris-hydrochloride (pH 6.8), 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 2 mM phenylmethyl sulfonylfluoride, and 10% (vol/vol) glycerol, and were heated at 100°C for 2 min. The SDS-polyacrylamide gel system used was that of Laemmli and Maizel, described by Laemmli (16), and electrophoresis was performed according to O'Farrell et al. (24). Radiolabeled protein bands on the gel were detected by fluorography (17).
RESULTS Identification of Ad2+ND2-specific polypeptides. Earlier studies have shown that Ad2 has the ability to inhibit host protein synthesis upon infection of permissive cells (1, 28). When human KB cells that had been infected with Ad2 for 24 h were pulsed with [35S]methionine for 10 min and the lysate from these labeled cells was analyzed on SDS-polyacrylamide gels, virtually all of the radiolabel was found incorporated into Ad2-specific polypeptides (Fig. la). Parallel cells infected with Ad2+ND2, in addition to synthesizing all of the Ad2-specific polypeptides present in Ad2-infected cells, also synthesized two new proteins with apparent molecular weights of 56,000 and 42,000 when analyzed on a 12.5% gel (Fig. lb) (32). Intracellular distribution of viral-specific proteins. To avoid selective loss of polypeptides with rapid turnover rates, infected cells were pulse-labeled with [35S]methionine for 20 min at 24 h postinfection and harvested immediately; the radiolabel was not chased with unlabeled methionine. The nuclear, cytoplasmic, and plasma membrane fractions were isolated, and parallel fractions from Ad2- and Ad2+ND2infected KB cells were analyzed on a 9% gel (Fig. lc through h). The apparently ubiquitous distribution of certain Ad2 structural proteins, including polypeptides II, Ha, V, and pVI (samples c through h), is consistent with the hypothesis that shortly after their synthesis in the cytoplasm, some of the viral polypeptides are assembled into structural units and rapidly transported to the nucleus for assembly of virions (10, 31). In contrast, polypeptides III and
694
J. VIROL.
JAY ET AL.
$
S~_-
kDal
I
0
,}
>1
$ -
qI u
1
I
r~ 120
h
100
Ufla
676.
ma
42
b\\
w
-
26 e~~~~af A
56K
0,-f
21
A
12
\ fl\e
L PW __ \
._I
--42K
C
e_ C d e f a b h FIG. 1. Fluorogram of SDS-polyacrylamide gels displaying proteins found in various subcellular fractions from KB cells infected with either Ad2 (a, c, e, g, and i) orAd2+ND2 (b, d, f, h, andj). Portions of each fraction containing 10,000 cpm were analyzed by electrophoresis on either a 12.5% (a and b) or a 9.0% (c through j) gel, as described in Materials and Methods. The amount of material from each of the four different subcellular fractions that was loaded onto the gel represented an enrichment of onefold (c and d), threefold (e and f), twofold (g and h), and fourfold (i and j), respectively. The exposure time was 2 days.
IV, which form the fiber and penton base, reside predominantly in the cytoplasmic fraction (samples c and d) and are apparently absent from the nuclei (samples g and h), whereas polypeptide pVII is found virtually quantitatively in the nuclear fraction (samples g and h). In addition to Ad2 structural proteins, isolated nuclei also contained, exclusively, four major Ad2 nonviron polypeptides with apparent molecular weights of 95,000, 82,500, 71,000, and 44,000. The latter two polypeptides are presumably the Ad2-specific DNA binding proteins (30). With regard to the SV40-specific proteins induced by Ad2+ND2, both the 56K and the 42K proteins were found in the cytoplasm (sample d) as well as in the plasma membrane fraction (sample f). Barely detectable amounts were associated with nuclei (sample h). When isolated
nuclei were first washed with a buffer containing 1% Triton X-100 to remove the nuclear membrane-associated ribosomes and any remaining cytoplasm that could not be stripped by mechanical means, the small fraction of 56K and 42K proteins previously found associated with isolated nuclei was now found in the nuclear wash (sample j). The nonionic detergent treatment also partially removed other proteins like polypeptides II and IIIa, but did not affect a significant percentage of the nonvirion, nuclear-specific polypeptides (samples i and j). Upon further fractionation of the cytoplasmic fraction by centrifugation at 100,000 x g, both the 56K and 42K proteins were found in the supernatant fraction (Fig. 2a and b) as well as in the pellet fraction (Fig. 2c and d). Of particular interest was the asymmetrical distribution of the two Ad2+ND2-induced SV40 proteins in
VOL. 23, 1977
SV40-SPECIFIC RIBOSOME-BINDING PROTEINS
the various subcellular fractions. The 100,000 x g pellet (samples c and d), like the whole-cell lysate (Fig. la and b), contained an equivalent amount of 42K protein and 56K protein, as indicated by the amount of radioactivity associated with each of the two bands on the gel. On the other hand, there was at least twice as much 42K protein as there was 56K protein in the 100,000 x g supernatant (samples a and b), whereas the reverse is true with the plasma membrane fraction, which contained two to three times more 56K protein than 42K protein cytoplasmic 100,000 x g supernatant
695
I
24-
E
in
Cl)
z Qu
0 it U)
plasma
membranes
pellet
FRACTION NUMBER
I, "~ ~ ~ 4 . -~~~Iij 3-~~~~56K o
a
b
c
d
e
-42K
f
FIG. 2. Fluorogram of a 12.5% SDS-polyacrylamide gel displaying differences in subcellular distribution between the two Ad2+ND2-specific proteins (b, d, and e). The amount of material from the 100,000 x g pellet that was layered onto the gel (c and d) represented approximately a fivefold enrichment with respect to either the 100,000 x g supernatant (a and b) or the plasma membranes (e and f) . Positions of the 56K and 42K proteins on the gel were identified by comparison with parallel fractions from Ad2+ND,-infected cells (a, c, and e), which did not induce these SV40-specific proteins. (Ad2+ND,, the nondefective Ad2-SV40 hybrid containing SV40 DNA segment 0.11 to 0.28 [21], has been found to induce only one new SV40-specific protein of 28K molecular weight [32]. The exposure time was 2 days.
FIG. 3. Velocity gradient analysis of ribosomes from Ad2+NDrinfected cells. (a) Cytoplasmic 100,000 x g pellet, containing ribosomes and Ad2specific structural units, was fractionated on a 10 to 40% (wtlwt) sucrose gradient, as described in Materials and Methods. (b) 40S region from the sucrose gradient shown in (a) was pooled, and portions were further fractionated separately on 10 to 40% (wtlwt) sucrose gradients made up in a buffer containing either 0.05 M (filled circles) or 0.50 M KCI (open circles), as described in Materials and Methods.
(samples e and f). With densitometric tracings of fluorograms, it can be calculated that in a 20min pulse, about 20% of each of the 42K and 56K proteins was present in a "complexed" form, which could be sedimented at 100,000 x g, in the cytoplasmic fraction. The 56K protein has a high affinity for the plasma membrane, with about 65% localized in that subcellular fraction and 15% found free in the cytoplasm. In contrast, only about 35% of the 42K protein was found associated with the plasma membrane, whereas 45% was found free in the cytosol. However, such quantitation does not take into account the possibility of selective loss of specific proteins during the fractionation and washing procedures. Isolation of ribosome-associated complexes. For further analysis of the complexed cytoplasmic forms of the SV40-specific proteins induced by Ad2+ND2, the 100,000 x g pellet from the cytoplasm was suspended again and fractionated on a 10 to 40% sucrose gradient (Fig. 3a). Determination of the trichloroacetic acid-precipitable radioactivity across the gradient indicated that a majority of the labeled proteins sedimented at about 11S (peak B), and a lesser amount remained virtually at the top of the gradient at about 6S (peak A). In addition, there were two minor components, one at 40S (peak C) and the other at 60S (peak D), cosedi-
696
J. VIROL.
JAY ET AL.
Salt
Low
; OIasri 1PrTi
!. :. .:-.
High Salt
Dqek Frac-tions LX B C: .: N
C
--42K
-.
41 K
-30K 28K
-s
164K
15K
ab
~
f
qh
4. Fluorogram of a 12.5% SDS-polyacrylgel displaying proteins from the peak fractions of the sucrose gradients used for the purification of ribosomes from Ad2+NDrinfected cells. Equal portions of the appropriate fractions from the sucrose gradients (Fig. 3b) were analyzed. Samples c, d, and e represented the peak fractions, designated A, B, and C, from the low-salt gradient (Fig. 3b, filed circles), and samples f, g, and h represented the corresponding fractions from the high-salt gradient (Fig. 3b, open circles). Sample i was a control from Ad2-infected cells, and it was equivalent to sample h from Ad2+ND2-infected cells. The cytoplasmic fraction from Ad.2ND2-infected cells (sample b) was used as a marker to identify the positions of the 56K and 42K proteins, which were not found in a parallel fraction from Ad2 +ND,-infected cells (sample a). The exposure time was 4 days.
FIG.
amide
menting with the two ribosomal subunits by the optical density scan at 254 nm. Analysis of the different fractions on SDS-polyacrylamide gels (data not shown) indicated that peak A was highly enriched for polypeptides IV and V, while peak B contained predominantly polypeptides II, Ha, III, and pVI. This observation agreed well with the previous finding that shortly after synthesis, polypeptides II, III, and IV were separately assembled in the cytoplasm into structural units of 12S, 10.5S, and 6S, reshown
spectively (10, 31). At least 80% of the 56K protein and 50% of the 42K protein were found sedimenting at the 40S region (peak C), but virtually none was found at 60S (peak D). The remainders of the 56K and 42K proteins were found at the top of the gradient (peak A). In an attempt to identify the true 40S ribosome-associated components, the material at the 40S region of the gradient (peak C) was pooled and fractionated on a second 10 to 40% sucrose gradient. As seen in Fig. 3b, only about half of the radioactivity was conserved in the 40S region (peak C); the rest of the material, sedimenting at 6S (peak A) and 11S (peak B), was well resolved from the 40S peak. Analysis of fractions on an SDS-polyacrylamide gel showed that all of the radioactivity in peaks A and B came from the large structural proteins, polypeptides II, III, and IV (Fig. 4c and d), which trailed into the 40S region during the first sucrose gradient separation (Fig. 3a). These polypeptides were, therefore, not true ribosome-associated proteins. The 56K and 42K proteins were not found at the top of the gradient where free proteins sediment, but they were found to reside quantitatively in peak C (Fig. 4e), indicating that all of the 56K and 42K proteins found in the 40S region of the first sucrose gradient are quantitatively associated with the 40S ribosomal subunits. The 40S subunits also contained a series of proteins with molecular weights of 41K, 30K, 28K, 16.4K, and 15K, which were detected only after enrichment for 40S ribosomes. Since they were also found in mock-infected cells (data not shown), these proteins must represent host ribosome-associated proteins that have continued to be synthesized, although in small quantities, under conditions where synthesis of most of the other cellular proteins has been shut off by the infecting virus. Though the amount of SV40-specific Ad2+ND2 proteins found associated with the 40S subunits in vivo may seem low relative to the total amount present in the cell, it should be noted that with a short radioisotope pulse, one can label only those ribosome-associated proteins with a rapid turnover rate, in contrast to virion polypeptides, which are synthesized continuously and in great excess. The turnover rate of a specific protein in association with ribosomes may be much lower than that of free protein. In fact, only a few of the 40S ribosomespecific proteins were detected under our labeling conditions. To exclude the possibility that the 56K and 42K proteins were contaminants of the 40S ribosomes, the pooled 40S material from the first
VOL. 23, 1977
SV40-SPECIFIC RIBOSOME-BINDING PROTEINS
697
sucrose gradient was dialyzed against a buffer ment with high concentrations of the monovacontaining 0.5 M KCl and then fractionated on lent cation. The slight decrease can be exa second sucrose gradient containing the same plained by the width of the protein band on the high-salt buffer. As seen in Fig. 3b, some of the slab gel being slightly greater in the presence radioactivity previously found associated with of high salt. That the 56K and 42K proteins the 40S subunits in the absence of the high-salt were indeed the SV40-specific polypeptides intreatment (Fig. 3b) was now found at the top of duced by Ad2+ND2 was confirmed by comparithe gradient. This radioactivity, however, rep- son with a parallel fraction from Ad2-infected resented not the release of Ad2+ND2-specific cells, which contained an identical distribution components (cf. e and h, Fig. 4), but rather the of all of the true 40S subunit-associated prorelease of a major proportion of the host-specific teins with molecular weights of 41K, 30K, 28K, ribosome-associated proteins (7). The 56K and 16.4K, and 15K, but not the two components in 42K proteins remained on the 40S ribosomal question (cf. h and i, Fig. 4). In vitro binding of viral proteins to ribosubunits and were almost unaffected by treatsomes. Since it may be argued that the apparently tight association of the 56K and 42K proPeak fractions teins with the 40S ribosomes could be an artifact generated during the fractionation and puA B C ri1 rification procedures, we have studied the ability of these SV40-specific proteins to bind to purified 40S ribosomal subunits in vitro. When free, 35S-labeled 56K and 42K proteins, present in the cytoplasmic 100,000 x g supernatant, were incubated with purified unlabeled 40S ribosomal subunits and the reaction products were analyzed on a 10 to 40% sucrose gradient, DI "~ a radioactivity profile very similar to that of Fig. 3a was obtained (data not shown). As expected, peak A (Fig. 5c) was found to contain ma-a the small structural proteins, including polypeptide V, whereas peak B (Fig. 5d) contained v- ,_ the larger proteins such as polypeptides II, III, and IV. Analysis of the 40S peak (Fig. 5e) dem56K onstrated the presence of two predominant _z components that coelectrophoresed with the 56K and 42K proteins found in the input mateS w - 42K rial from Ad2+ND2-infected cells (Fig. 5b), but not present in a parallel sample from Ad2-infected cells (Fig. 5a). It cannot be argued that the presence of these SV40-specific proteins on the 40S subunits was due to contaminating ribosomes present in the 35S-labeled 100,000 x g supernatant, since, in the absence of added 40S subunits, the 56K and 42K proteins were found in the top of the graa b c d e dient and not in the 40S region (data not FIG. 5. Fluorogram of a 9.0% SDS-polyacryl- shown). Furthermore, if the 56K and 42K proamide gel displaying proteins from the peak fractions teins found associated with the 40S subunits of a sucrose gradient used to show the binding of the were explained by contamination with 40S subAd2+ND2specific proteins to 40S subunits in vitro. units labeled in vivo, one would have expected The cytoplasmic 100,000 x g supernatant from to see the low-molecular-weight proteins menAd2+ND2infected cells (b), containing the 56K and tioned earlier (41K, 30K, 28K, 16.4K, and 15K) 42K proteins that were not present in a parallel frac- migrating ahead of the SV40-specific proteins tion from Ad2-infected cells (a), was incubated with 4e and h); none can be detected. In purified 40S subunits, and the reaction mixture was (see Fig. no other labeled protein, apart fact, virtually analyzed on a 10 to 40% sucrose gradient, as described in Materials and Methods. Samples c, d, and from the 56K and 42K polypeptides, is found associated with the 40S subunits. e represented the peak fractions, corresponding to A, Under the conditions used, the binding of the B, and C in Fig. 3a, taken from the sucrose gradient. 42K protein was quantitative; all of the 42K The exposure time was 3 days.
698
JAY ET AL.
J. VIROL.
(data not shown), suggests that at least one of these Ad2+ND2 proteins may also be responsible for the SV40 U antigen activity. We have also found, as was previously reported (5), that the 56K and 42K proteins can be specifically immunoprecipitated with sera obtained from hamsters bearing SV40-induced tumors (data not shown). Whether these SV40specific Ad2+ND2 proteins do in fact share antigenic determinants with SV40 T antigen, U antigen, or both is presently being studied. Our finding that these Ad2+ND2 proteins are absent DISCUSSION from detergent-washed nuclei varies with an The Ad2-SV40 hybrid virus, Ad2+ND2, differs earlier report that as much as 50% of both from wild-type Ad2 in that part of the early proteins are found in the nuclear fraction alone region of SV40 DNA has been integrated into (5). the Ad2 genome (18). As a result, Ad2+ND2 While it is difficult to correlate biological codes for two new polypeptides (32), apparently functions with the various SV40-specific immuSV40-specific and with molecular weights of nological activities, an SV40-induced function 42K and 56K. These polypeptides have been has been implicated in the enhancement of the shown to possess extensive amino acid se- growth of human adenoviruses in simian cells quence homology (5). Ad2+ND2 has previously (26). This helper function has been demonbeen shown to induce two of the three SV40- strated to be SV40-coded (9, 11, 13), and the specific, early immunological activities that are SV40 fragment present in Ad2+ND2 is sufficient found in SV40-infected and transformed cells to code for this function (19). In the absence of (19): U antigen, detected by complement fixa- SV40 enhancement, synthesis of late Ad2-spetion and immunofluorescent methods, and tu- cific structural proteins is either completely or mor-specific transplantation antigen, sepa- partially blocked (2, 15), despite the presence of rately detected by in vivo immunological as- their corresponding mRNA's (6, 15). This obsersays. vation has led to the hypothesis that the SV40We have found that significant amounts of enhancing component functions by alleviating the two SV40-specific proteins induced by the translational block, perhaps by providing Ad2+ND2, and detected after a short pulse of ribosome specificity for mRNA recognition (22, 35S-labeled methionine, are localized on the 23), or, alternatively, by blocking the activity of plasma membrane (65% of the 56K protein and a ribosome-associated and mRNA-specific 35% of the 42K protein). This distribution did RNase (15). Our findings that a significant fracnot seem to change significantly either when tion of each of the two SV40-specific Ad2+ND2the labeling time was extended to 2 h, or when induced proteins is found tightly associated the short pulse was subsequently chased with with 40S ribosomal subunits, and that the 56K unlabeled methionine for 60 min (data not and 42K proteins found free in the cytoplasm shown). Our finding suggests that, in this loca- can bind preferentially and almost quantitation, at least one of these proteins may be the tively to 40S subunits in vitro, lend support to tumor-specific transplantation antigen, which the proposal that the SV40-specific, Ad2+ND2by definition is a cell-surface antigen (29). induced proteins may indeed function directly Moreover, we have demonstrated in other stud- as translational control elements in cells. We ies that mice preimmunized with these believe that the use of purified Ad2+ND2 proAd2+ND2-induced proteins do not support tu- teins in a well-characterized cell-free proteinmor formation upon subsequent challenge with synthesizing system will lead to better underSV40 tumor cells (G. Jay et al., unpublished standing of the molecular basis for SV40 endata). hancement, and, hence, the function of the reTogether with the earlier finding, by immu- sponsible SV40 antigen. nofluorescent staining, that U antigen has a ACKNOWLEDGMENTS diffuse cytoplasmic distribution in Ad2+ND2We thank Barrie J. Carter and Heiner Westphal for their from the nucleus infected cells and is absent (19), our observation that both of the SV40- valuable comments on the manuscript. specific Ad2+ND2 proteins are found in the cytoLITERATURE CITED plasm and not in the nucleus, even after contin- 1. Anderson, C. W., S. G. Baum, and R. F. Gesteland. uous labeling for 2 h or when the short radioac1973. Processing of adenovirus 2-induced proteins. J. Virol. 12:241-252. tive pulse was subsequently chased for 1 h
protein was found associated with the 40S ribosomes, with none located at the top of the gradient where unbound proteins have been shown to remain in the absence of added 40S ribosomes. Because of the presence of other proteins that migrated in the vicinity of the 56K protein (Fig. 5a and b), it was difficult to determine the extent of binding of the 56K protein, except to conclude that no less than 60% was associated with the 40S subunits.
VOL. 23, 1977
SV40-SPECIFIC RIBOSOME-BINDING PROTEINS
2. Baum, S. G., M. S. Horwitz, and J. V. Maizel, Jr. 1972. Studies of the mechanism of enhancement of human adenovirus infection in monkey cells by simian virus 40. J. Virol. 10:211-219. 3. Black, P. H., W. P. Rowe, H. C. Turner, and R. J. Huebner. 1963. A specific complement-fixing antigen present in SV40 tumor and transformed cells. Proc. Natl. Acad. Sci. U.S.A. 50:1148-1156. 4. Brunette, D. M., and J. E. Till. 1971. A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membr. Biol. 5:215-224. 5. Deppert, W., and G. Walter. 1976. Simian virus 40 (SV40) tumor-specific proteins in nucleus and plasma membrane of HeLa cells infected by adenovirus 2SV40 hybrid virus Ad2+ND2. Proc. Natl. Acad. Sci. U.S.A. 73:2505-2509. 6. Fox, R. I., and S. G. Baum. 1972. Synthesis of viral ribonucleic acid during restricted adenovirus infection. J. Virol. 10:220-227. 7. Freienstein, C., and G. Blobel. 1975. Nonribosomal proteins associated with eukaryotic native small ribosomal subunits. Proc. Natl. Acad. Sci. U.S.A. 72:3392-3396. 8. Girardi, A. J., and V. Defendi. 1970. Induction of SV40 transplantation antigen (TrAg) during the lytic cycle. Virology 42:688-698. 9. Grodzicker, T., C. Anderson, P. A. Sharp, and J. Sambrook. 1974. Conditional lethal mutants of adenovirus 2-simian virus 40 hybrids. I. Host range mutants of Ad2+ND,. J. Virol. 13:1237-1244. 10. Horwitz, M. S., M. D. Scharff, and J. V. Maizel, Jr. 1969. Synthesis and assembly of adenovirus 2. I. Polypeptide synthesis, assembly of capsomeres, and morphogenesis of the virion. Virology 39:682-694. 11. Jerkofsky, M. 1975. Enhancement of the replication of human adenovirus in simian cells by a series of temperature-sensitive mutants of simian virus 40. Virology 65:579-582. 12. Khera, K. S., A. Ashkenazi, F. Rapp, and J. L. Melnick. 1963. Immunity in hamsters to cells transformed in vitro and in vivo by SV40. J. Immunol. 91:604-613. 13. Kimura, G. 1974. Genetic evidence for SV40 gene function in enhancement of replication of human adenovirus in simian cells. Nature (London) 248:590-592. 14. Kit, S. 1968. Viral-induced enzymes and the problem of viral oncogenesis. Adv. Cancer Res. 11:73-221. 15. Klessig, D. F., and C. W. Anderson. 1975. Block to multiplication of adenovirus serotype 2 in monkey cells. J. Virol. 16:1650-1668. 16. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 17. Laskey, R. A., and A. D. Mills. 1975. Quantitative film detection of 3H and '4C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56:335-341.
699
18. Levine, A. S., M. J. Levin, M. N. Oxman, and A. M. Lewis, Jr. 1973. Studies of nondefective adenovirus 2simian virus 40 hybrid viruses. VII. Characterization of the simian virus 40 RNA species induced by five nondefective hybrid viruses. J. Virol. 11:672-681. 19. Lewis, A. M., Jr., A. S. Levine, C. S. Crumpacker, M. J. Levin, R. J. Samaha, and P. H. Henry. 1973. Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. V. Isolation of additional hybrids which differ in their simian virus 40-specific biological properties. J. Virol. 11:655-664. 20. Lewis, A. M., Jr., and W. P. Rowe. 1971. Studies on nondefective adenovirus-simian virus 40 hybrid viruses. I. A newly characterized simian virus 40 antigen induced by the Ad2+ND, virus. J. Virol. 7:189197. 21. Morrow, J. F., P. Berg, T. J. Kelly, Jr., and A. M. Lewis, Jr. 1973. Mapping of simian virus 40 early functions on the viral chromosome. J. Virol. 12:653658. 22. Nakajima, K., H. Ishitsuka, and K. Oda. 1974. An SV40 induced initiation factor for protein synthesis concerned with the regulation of permissiveness. Nature (London) 252:649-653. 23. Nakajima, K., and K. Oda. 1975. The alteration of ribosomes for mRNA selection concerned with adenovirus growth in SV40-infected simian cells. Virology 67:85-93. 24. O'Farrell, P. Z., L. M. Gold, and W. H. Huang. 1973. The identification of prereplicative bacteriophage T4 proteins. J. Biol. Chem. 248:5499-5501. 25. Pettersson, U., and J. Sambrook. 1973. Amount of viral DNA in the genome of cells transformed by adenovirus type 2. J. Mol. Biol. 73:125-130. 26. Rabson, A. S., G. T. O'Conor, I. K. Berezesky, and F. J. Paul. 1964. Enhancement of adenovirus growth in African green monkey kidney cell cultures by SV40. Proc. Soc. Exp. Biol. Med. 116:187-190. 27. Rapp, F., T. Kitahara, J. S. Butel, and J. L. Melnick. 1964. Synthesis of SV40 tumor antigen during replication of simian papovavirus (SV40). Proc. Natl. Acad. Sci. U.S.A. 52:1138-1142. 28. Russell, W. C., and J. J. Skehel. 1972. The polypeptides of adenovirus-infected cells. J. Gen. Virol. 15:45-57. 29. Tevethia, S. S., M. Katz, and F. Rapp. 1965. New surface antigen in cells transformed by simian papovavirus SV40. Proc. Soc. Exp. Biol. Med. 119:896-901. 30. Van der Vliet, P. C., and A. J. Levine. 1973. DNAbinding proteins specific for cells infected by adenovirus. Nature (London) New Biol. 246:170-174. 31. Velicer, L., and H. S. Ginsberg. 1970. Synthesis, transport, and morphogenesis of type 5 adenovirus capsid proteins. J. Virol. 5:338-352. 32. Walter, G., and H. Martin. 1975. Simian virus 40-specific proteins in HeLa cells infected with nondefective adenovirus 2-simian virus 40 hybrid viruses. J. Virol. 16:1236-1247.
Vol. 23, No. 3 Printed in U.S.A.
Simian Virus 40-Specific Ribosome-Binding Proteins Induced by a Nondefective Adenovirus 2-Simian Virus 40 Hybrid GILBERT JAY,* FRANCIS T. JAY, ROBERT M. FRIEDMAN, AND ARTHUR S. LEVINE Pediatric Oncology Branch, National Cancer Institute, and Laboratory of Experimental Pathology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received for publication 3 March 1977
We have studied the intracellular distribution of the two simian virus 40specific proteins, with apparent molecular weights of 56,000 and 42,000, detectable in human KB cells infected by a nondefective adenovirus 2-simian virus 40 hybrid, Ad2+ND2. After a 20-min pulse of ['5S]methionine, about two-thirds of the newly synthesized 56K protein and one-third of the 42K protein were found localized on the plasma membrane. The remainder of each protein was found in the cytoplasm, whereas the nuclear fraction was virtually free of either component. A significant portion of both proteins present in the cytoplasmic fraction was completed to the 40S ribosomal subunits and was not removed by treatment with 0.5 M KCl. Moreover, the portion that was found free in the cytoplasm could bind preferentially and quantitatively to purified 40S ribosomes in vitro, leading us to propose that these simian virus 40 proteins may act as translational control elements in cells.
Simian virus 40 (SV40) induces three dis- distribution of the SV40-specific proteins syntinct immunological activities detectable in thesized by one of the nondefective hybrids, either transformed or productively infected Ad2+ND2 (which contains the SV40 DNA segcells: T antigen (3, 27), tumor-specific trans- ment 0.11 to 0.44 map unit from the endonuplantation antigen (8, 12), and U antigen (20). clease R-EcoRI cleavage site), with the hope The relationship between each of these anti- that knowledge about their "functional" localgens and their exact function in cells remain ization may permit the design of cell-free sysspeculative. tems that would elucidate the specific in vivo Study of these early SV40 antigens is techni- function of the early SV40 antigens. cally difficult because SV40 not only fails to MATERIALS AND METHODS inhibit host protein synthesis upon infection, but actually stimulates certain host cell biosynViruses. Ad2, Ad2+ND,, and Ad2+ND2 stocks thetic activities (14). Consequently, the amount were grown in monolayer cultures of human KB of early viral antigens present at any time after cells, infected at a multiplicity of 5 PFU/cell, and infection is quite small compared with the purified by twice banding on CsCl gradients after background of newly synthesized host proteins; extraction from cell sonic extracts with Freon 113 therefore, their purification has not been (25). The stock viruses were plaque titrated on monolayers of primary human embryonic kidney cells achieved. The nondefective adenovirus 2 (Ad2)-SV40 (19). and labeling of cells. Human KB cells hybrid viruses appear to form an ideal system in Infection monolayers were infected with purified Ad2, for study of the function of various SV40 early Ad2+ND,, or Ad2+ND2 at a multiplicity of 20 PFU/ antigens. These hybrid viruses contain differ- cell. Twenty-four hours postinfection, the monolayent amounts of SV40 DNA covalently inserted ers were rinsed and incubated at 370C in Earle at a unique site in the Ad2 genome (21). As a balanced salt solution containing 5% minimum esresult, several of these hybrid viruses have ac- sential medium, 2% fetal calf serum, and 40 ,uCi of Lquired the genetic information needed to code [35S]methionine per ml (300 to 400 Ci/mmol). After 10 to 20 min, the cells were rinsed with phosphatefor the various early SV40-specific immunologi- buffered saline and scraped from the bottles. cal activities (19). In contrast to SV40 infection, Subcellular fractionation. Washed cells were sushybrid virus infection inhibits host protein syn- pended in a buffer 10 mM Tris-hydrothesis, maximizing the opportunity to isolate chloride (pH 7.4), 10containing mM KCl, 2 mM MgCl2, and 2 SV40-specific proteins. mM dithiothreitol, and were homogenized with a We have attempted to study the intracellular glass Dounce tissue grinder until more than 95% of 692
VOL. 23, 1977
SV40-SPECIFIC RIBOSOME-BINDING PROTEINS
the cells were seen to be broken under the phasecontrast microscope (eight strokes). The cell lysate was spun at 800 x g for 15 min, and both the supernatant and the pellet were saved for further fractionation. (i) Plasma membranes. The surface membranes and nuclei in the pellet from the low-speed centrifugation were separated by the dextran-polyethylene glycol two-phase system as described by Brunette and Till (4), except that 10 mM NaCl was present instead of ZnCl2. Upon separation of the two phases by centrifugation at 11,700 x g for 10 min, the membranes were found at the interphase. The nuclear pellet was saved for subsequent fractionation, while the membrane fraction was further purified as described (4). Purified membranes were completely free of nuclei when examined by phase-contrast microscopy. (ii) Nuclei and nuclear wash. The nuclear pellet from the first two-phase separation was again suspended in equal volumes of the two phases by homogenization in a Dounce tissue grinder. Any contaminating surface membrane was separated from the nuclei upon phase separation by centrifugation. The nuclear pellet was suspended in a buffer composed of 10 mM Tris-hydrochloride (pH 6.8), 150 mM KCl, 5 mM MgCl2, and 1% Triton X-100, again by homogenization in a Dounce tissue grinder. After 10 min at 4VC, the washed nuclei were separated from the nuclear wash by centrifugation. Suspended nuclei remained fully intact and free of membranous material as visualized by phase-contrast microscopy.
(iii) Cytoplasmic fractions. The supernatant from the initial low-speed centrifugation of the cell homogenate was further centrifuged for 30 min at 30,000 x g. The resultant supernatant, containing the cytoplasmic fraction, was further fractionated by centrifugation for 4 h at 100,000 x g. The resultant supernatant was saved and the pellet was suspended by stirring in a low-salt buffer (buffer A) containing 10 mM Tris-hydrochloride (pH 7.4), 50 mM KCl, 2 mM MgCl2, and 2 mM dithiothreitol. Ribosomal subunits. The suspended 100,000 x g pellet containing the ribosomes was fractionated on 10 to 40% (wt/wt) sucrose gradients made up in buffer A. About 25 A260 units were layered onto each 12.5-ml gradient, and centrifugation was for 5 h at 40,000 rpm (284,000 x g) in a Spinco SW40 rotor. The gradients were collected with an ISCO gradient analyzer, and a portion from each fraction was analyzed for total trichloroacetic acid-precipitable radioactivity. The fractions corresponding to the 40S region were pooled. Half of the pooled fraction was dialyzed against buffer A and the other half against a highsalt buffer (the same as buffer A but containing 500 mM KCl). Both samples were concentrated by sprinkling Sephadex G-150 over the dialysis bag, and they were further purified on 12.5-ml 10 to 40% (wt/wt) sucrose gradients made up in the corresponding buffer. After centrifugation for 5 h at 40,000 rpm (284,000 x g), fractions were collected, and a portion from each fraction was assayed for radioactivity. Because of the change in conformation and the loss of significant amounts of ribosome-
693
associated protein factors induced by high-salt conditions, the 40S subunits appeared to sediment at a lower s value in the presence of high salt (see Fig. 3b). In vitro ribosome binding. The 100,000 x g supernatant was dialyzed against a buffer containing 50 mM Tris-hydrochloride (pH 7.4), 100 mM KCl, 2 mM MgCl2, and 2 mM dithiothreitol, and it was subsequently incubated at 37°C for 30 min with 1.0 A260 unit of 40S ribosomes, obtained from either mockor Ad2-infected cells and purified on sucrose gradients. The reaction products were analyzed on 10 to 40% (wt/wt) sucrose gradients made up in buffer A. Polyacrylamide gel electrophoresis. All samples for analysis contained 62 mM Tris-hydrochloride (pH 6.8), 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 2 mM phenylmethyl sulfonylfluoride, and 10% (vol/vol) glycerol, and were heated at 100°C for 2 min. The SDS-polyacrylamide gel system used was that of Laemmli and Maizel, described by Laemmli (16), and electrophoresis was performed according to O'Farrell et al. (24). Radiolabeled protein bands on the gel were detected by fluorography (17).
RESULTS Identification of Ad2+ND2-specific polypeptides. Earlier studies have shown that Ad2 has the ability to inhibit host protein synthesis upon infection of permissive cells (1, 28). When human KB cells that had been infected with Ad2 for 24 h were pulsed with [35S]methionine for 10 min and the lysate from these labeled cells was analyzed on SDS-polyacrylamide gels, virtually all of the radiolabel was found incorporated into Ad2-specific polypeptides (Fig. la). Parallel cells infected with Ad2+ND2, in addition to synthesizing all of the Ad2-specific polypeptides present in Ad2-infected cells, also synthesized two new proteins with apparent molecular weights of 56,000 and 42,000 when analyzed on a 12.5% gel (Fig. lb) (32). Intracellular distribution of viral-specific proteins. To avoid selective loss of polypeptides with rapid turnover rates, infected cells were pulse-labeled with [35S]methionine for 20 min at 24 h postinfection and harvested immediately; the radiolabel was not chased with unlabeled methionine. The nuclear, cytoplasmic, and plasma membrane fractions were isolated, and parallel fractions from Ad2- and Ad2+ND2infected KB cells were analyzed on a 9% gel (Fig. lc through h). The apparently ubiquitous distribution of certain Ad2 structural proteins, including polypeptides II, Ha, V, and pVI (samples c through h), is consistent with the hypothesis that shortly after their synthesis in the cytoplasm, some of the viral polypeptides are assembled into structural units and rapidly transported to the nucleus for assembly of virions (10, 31). In contrast, polypeptides III and
694
J. VIROL.
JAY ET AL.
$
S~_-
kDal
I
0
,}
>1
$ -
qI u
1
I
r~ 120
h
100
Ufla
676.
ma
42
b\\
w
-
26 e~~~~af A
56K
0,-f
21
A
12
\ fl\e
L PW __ \
._I
--42K
C
e_ C d e f a b h FIG. 1. Fluorogram of SDS-polyacrylamide gels displaying proteins found in various subcellular fractions from KB cells infected with either Ad2 (a, c, e, g, and i) orAd2+ND2 (b, d, f, h, andj). Portions of each fraction containing 10,000 cpm were analyzed by electrophoresis on either a 12.5% (a and b) or a 9.0% (c through j) gel, as described in Materials and Methods. The amount of material from each of the four different subcellular fractions that was loaded onto the gel represented an enrichment of onefold (c and d), threefold (e and f), twofold (g and h), and fourfold (i and j), respectively. The exposure time was 2 days.
IV, which form the fiber and penton base, reside predominantly in the cytoplasmic fraction (samples c and d) and are apparently absent from the nuclei (samples g and h), whereas polypeptide pVII is found virtually quantitatively in the nuclear fraction (samples g and h). In addition to Ad2 structural proteins, isolated nuclei also contained, exclusively, four major Ad2 nonviron polypeptides with apparent molecular weights of 95,000, 82,500, 71,000, and 44,000. The latter two polypeptides are presumably the Ad2-specific DNA binding proteins (30). With regard to the SV40-specific proteins induced by Ad2+ND2, both the 56K and the 42K proteins were found in the cytoplasm (sample d) as well as in the plasma membrane fraction (sample f). Barely detectable amounts were associated with nuclei (sample h). When isolated
nuclei were first washed with a buffer containing 1% Triton X-100 to remove the nuclear membrane-associated ribosomes and any remaining cytoplasm that could not be stripped by mechanical means, the small fraction of 56K and 42K proteins previously found associated with isolated nuclei was now found in the nuclear wash (sample j). The nonionic detergent treatment also partially removed other proteins like polypeptides II and IIIa, but did not affect a significant percentage of the nonvirion, nuclear-specific polypeptides (samples i and j). Upon further fractionation of the cytoplasmic fraction by centrifugation at 100,000 x g, both the 56K and 42K proteins were found in the supernatant fraction (Fig. 2a and b) as well as in the pellet fraction (Fig. 2c and d). Of particular interest was the asymmetrical distribution of the two Ad2+ND2-induced SV40 proteins in
VOL. 23, 1977
SV40-SPECIFIC RIBOSOME-BINDING PROTEINS
the various subcellular fractions. The 100,000 x g pellet (samples c and d), like the whole-cell lysate (Fig. la and b), contained an equivalent amount of 42K protein and 56K protein, as indicated by the amount of radioactivity associated with each of the two bands on the gel. On the other hand, there was at least twice as much 42K protein as there was 56K protein in the 100,000 x g supernatant (samples a and b), whereas the reverse is true with the plasma membrane fraction, which contained two to three times more 56K protein than 42K protein cytoplasmic 100,000 x g supernatant
695
I
24-
E
in
Cl)
z Qu
0 it U)
plasma
membranes
pellet
FRACTION NUMBER
I, "~ ~ ~ 4 . -~~~Iij 3-~~~~56K o
a
b
c
d
e
-42K
f
FIG. 2. Fluorogram of a 12.5% SDS-polyacrylamide gel displaying differences in subcellular distribution between the two Ad2+ND2-specific proteins (b, d, and e). The amount of material from the 100,000 x g pellet that was layered onto the gel (c and d) represented approximately a fivefold enrichment with respect to either the 100,000 x g supernatant (a and b) or the plasma membranes (e and f) . Positions of the 56K and 42K proteins on the gel were identified by comparison with parallel fractions from Ad2+ND,-infected cells (a, c, and e), which did not induce these SV40-specific proteins. (Ad2+ND,, the nondefective Ad2-SV40 hybrid containing SV40 DNA segment 0.11 to 0.28 [21], has been found to induce only one new SV40-specific protein of 28K molecular weight [32]. The exposure time was 2 days.
FIG. 3. Velocity gradient analysis of ribosomes from Ad2+NDrinfected cells. (a) Cytoplasmic 100,000 x g pellet, containing ribosomes and Ad2specific structural units, was fractionated on a 10 to 40% (wtlwt) sucrose gradient, as described in Materials and Methods. (b) 40S region from the sucrose gradient shown in (a) was pooled, and portions were further fractionated separately on 10 to 40% (wtlwt) sucrose gradients made up in a buffer containing either 0.05 M (filled circles) or 0.50 M KCI (open circles), as described in Materials and Methods.
(samples e and f). With densitometric tracings of fluorograms, it can be calculated that in a 20min pulse, about 20% of each of the 42K and 56K proteins was present in a "complexed" form, which could be sedimented at 100,000 x g, in the cytoplasmic fraction. The 56K protein has a high affinity for the plasma membrane, with about 65% localized in that subcellular fraction and 15% found free in the cytoplasm. In contrast, only about 35% of the 42K protein was found associated with the plasma membrane, whereas 45% was found free in the cytosol. However, such quantitation does not take into account the possibility of selective loss of specific proteins during the fractionation and washing procedures. Isolation of ribosome-associated complexes. For further analysis of the complexed cytoplasmic forms of the SV40-specific proteins induced by Ad2+ND2, the 100,000 x g pellet from the cytoplasm was suspended again and fractionated on a 10 to 40% sucrose gradient (Fig. 3a). Determination of the trichloroacetic acid-precipitable radioactivity across the gradient indicated that a majority of the labeled proteins sedimented at about 11S (peak B), and a lesser amount remained virtually at the top of the gradient at about 6S (peak A). In addition, there were two minor components, one at 40S (peak C) and the other at 60S (peak D), cosedi-
696
J. VIROL.
JAY ET AL.
Salt
Low
; OIasri 1PrTi
!. :. .:-.
High Salt
Dqek Frac-tions LX B C: .: N
C
--42K
-.
41 K
-30K 28K
-s
164K
15K
ab
~
f
qh
4. Fluorogram of a 12.5% SDS-polyacrylgel displaying proteins from the peak fractions of the sucrose gradients used for the purification of ribosomes from Ad2+NDrinfected cells. Equal portions of the appropriate fractions from the sucrose gradients (Fig. 3b) were analyzed. Samples c, d, and e represented the peak fractions, designated A, B, and C, from the low-salt gradient (Fig. 3b, filed circles), and samples f, g, and h represented the corresponding fractions from the high-salt gradient (Fig. 3b, open circles). Sample i was a control from Ad2-infected cells, and it was equivalent to sample h from Ad2+ND2-infected cells. The cytoplasmic fraction from Ad.2ND2-infected cells (sample b) was used as a marker to identify the positions of the 56K and 42K proteins, which were not found in a parallel fraction from Ad2 +ND,-infected cells (sample a). The exposure time was 4 days.
FIG.
amide
menting with the two ribosomal subunits by the optical density scan at 254 nm. Analysis of the different fractions on SDS-polyacrylamide gels (data not shown) indicated that peak A was highly enriched for polypeptides IV and V, while peak B contained predominantly polypeptides II, Ha, III, and pVI. This observation agreed well with the previous finding that shortly after synthesis, polypeptides II, III, and IV were separately assembled in the cytoplasm into structural units of 12S, 10.5S, and 6S, reshown
spectively (10, 31). At least 80% of the 56K protein and 50% of the 42K protein were found sedimenting at the 40S region (peak C), but virtually none was found at 60S (peak D). The remainders of the 56K and 42K proteins were found at the top of the gradient (peak A). In an attempt to identify the true 40S ribosome-associated components, the material at the 40S region of the gradient (peak C) was pooled and fractionated on a second 10 to 40% sucrose gradient. As seen in Fig. 3b, only about half of the radioactivity was conserved in the 40S region (peak C); the rest of the material, sedimenting at 6S (peak A) and 11S (peak B), was well resolved from the 40S peak. Analysis of fractions on an SDS-polyacrylamide gel showed that all of the radioactivity in peaks A and B came from the large structural proteins, polypeptides II, III, and IV (Fig. 4c and d), which trailed into the 40S region during the first sucrose gradient separation (Fig. 3a). These polypeptides were, therefore, not true ribosome-associated proteins. The 56K and 42K proteins were not found at the top of the gradient where free proteins sediment, but they were found to reside quantitatively in peak C (Fig. 4e), indicating that all of the 56K and 42K proteins found in the 40S region of the first sucrose gradient are quantitatively associated with the 40S ribosomal subunits. The 40S subunits also contained a series of proteins with molecular weights of 41K, 30K, 28K, 16.4K, and 15K, which were detected only after enrichment for 40S ribosomes. Since they were also found in mock-infected cells (data not shown), these proteins must represent host ribosome-associated proteins that have continued to be synthesized, although in small quantities, under conditions where synthesis of most of the other cellular proteins has been shut off by the infecting virus. Though the amount of SV40-specific Ad2+ND2 proteins found associated with the 40S subunits in vivo may seem low relative to the total amount present in the cell, it should be noted that with a short radioisotope pulse, one can label only those ribosome-associated proteins with a rapid turnover rate, in contrast to virion polypeptides, which are synthesized continuously and in great excess. The turnover rate of a specific protein in association with ribosomes may be much lower than that of free protein. In fact, only a few of the 40S ribosomespecific proteins were detected under our labeling conditions. To exclude the possibility that the 56K and 42K proteins were contaminants of the 40S ribosomes, the pooled 40S material from the first
VOL. 23, 1977
SV40-SPECIFIC RIBOSOME-BINDING PROTEINS
697
sucrose gradient was dialyzed against a buffer ment with high concentrations of the monovacontaining 0.5 M KCl and then fractionated on lent cation. The slight decrease can be exa second sucrose gradient containing the same plained by the width of the protein band on the high-salt buffer. As seen in Fig. 3b, some of the slab gel being slightly greater in the presence radioactivity previously found associated with of high salt. That the 56K and 42K proteins the 40S subunits in the absence of the high-salt were indeed the SV40-specific polypeptides intreatment (Fig. 3b) was now found at the top of duced by Ad2+ND2 was confirmed by comparithe gradient. This radioactivity, however, rep- son with a parallel fraction from Ad2-infected resented not the release of Ad2+ND2-specific cells, which contained an identical distribution components (cf. e and h, Fig. 4), but rather the of all of the true 40S subunit-associated prorelease of a major proportion of the host-specific teins with molecular weights of 41K, 30K, 28K, ribosome-associated proteins (7). The 56K and 16.4K, and 15K, but not the two components in 42K proteins remained on the 40S ribosomal question (cf. h and i, Fig. 4). In vitro binding of viral proteins to ribosubunits and were almost unaffected by treatsomes. Since it may be argued that the apparently tight association of the 56K and 42K proPeak fractions teins with the 40S ribosomes could be an artifact generated during the fractionation and puA B C ri1 rification procedures, we have studied the ability of these SV40-specific proteins to bind to purified 40S ribosomal subunits in vitro. When free, 35S-labeled 56K and 42K proteins, present in the cytoplasmic 100,000 x g supernatant, were incubated with purified unlabeled 40S ribosomal subunits and the reaction products were analyzed on a 10 to 40% sucrose gradient, DI "~ a radioactivity profile very similar to that of Fig. 3a was obtained (data not shown). As expected, peak A (Fig. 5c) was found to contain ma-a the small structural proteins, including polypeptide V, whereas peak B (Fig. 5d) contained v- ,_ the larger proteins such as polypeptides II, III, and IV. Analysis of the 40S peak (Fig. 5e) dem56K onstrated the presence of two predominant _z components that coelectrophoresed with the 56K and 42K proteins found in the input mateS w - 42K rial from Ad2+ND2-infected cells (Fig. 5b), but not present in a parallel sample from Ad2-infected cells (Fig. 5a). It cannot be argued that the presence of these SV40-specific proteins on the 40S subunits was due to contaminating ribosomes present in the 35S-labeled 100,000 x g supernatant, since, in the absence of added 40S subunits, the 56K and 42K proteins were found in the top of the graa b c d e dient and not in the 40S region (data not FIG. 5. Fluorogram of a 9.0% SDS-polyacryl- shown). Furthermore, if the 56K and 42K proamide gel displaying proteins from the peak fractions teins found associated with the 40S subunits of a sucrose gradient used to show the binding of the were explained by contamination with 40S subAd2+ND2specific proteins to 40S subunits in vitro. units labeled in vivo, one would have expected The cytoplasmic 100,000 x g supernatant from to see the low-molecular-weight proteins menAd2+ND2infected cells (b), containing the 56K and tioned earlier (41K, 30K, 28K, 16.4K, and 15K) 42K proteins that were not present in a parallel frac- migrating ahead of the SV40-specific proteins tion from Ad2-infected cells (a), was incubated with 4e and h); none can be detected. In purified 40S subunits, and the reaction mixture was (see Fig. no other labeled protein, apart fact, virtually analyzed on a 10 to 40% sucrose gradient, as described in Materials and Methods. Samples c, d, and from the 56K and 42K polypeptides, is found associated with the 40S subunits. e represented the peak fractions, corresponding to A, Under the conditions used, the binding of the B, and C in Fig. 3a, taken from the sucrose gradient. 42K protein was quantitative; all of the 42K The exposure time was 3 days.
698
JAY ET AL.
J. VIROL.
(data not shown), suggests that at least one of these Ad2+ND2 proteins may also be responsible for the SV40 U antigen activity. We have also found, as was previously reported (5), that the 56K and 42K proteins can be specifically immunoprecipitated with sera obtained from hamsters bearing SV40-induced tumors (data not shown). Whether these SV40specific Ad2+ND2 proteins do in fact share antigenic determinants with SV40 T antigen, U antigen, or both is presently being studied. Our finding that these Ad2+ND2 proteins are absent DISCUSSION from detergent-washed nuclei varies with an The Ad2-SV40 hybrid virus, Ad2+ND2, differs earlier report that as much as 50% of both from wild-type Ad2 in that part of the early proteins are found in the nuclear fraction alone region of SV40 DNA has been integrated into (5). the Ad2 genome (18). As a result, Ad2+ND2 While it is difficult to correlate biological codes for two new polypeptides (32), apparently functions with the various SV40-specific immuSV40-specific and with molecular weights of nological activities, an SV40-induced function 42K and 56K. These polypeptides have been has been implicated in the enhancement of the shown to possess extensive amino acid se- growth of human adenoviruses in simian cells quence homology (5). Ad2+ND2 has previously (26). This helper function has been demonbeen shown to induce two of the three SV40- strated to be SV40-coded (9, 11, 13), and the specific, early immunological activities that are SV40 fragment present in Ad2+ND2 is sufficient found in SV40-infected and transformed cells to code for this function (19). In the absence of (19): U antigen, detected by complement fixa- SV40 enhancement, synthesis of late Ad2-spetion and immunofluorescent methods, and tu- cific structural proteins is either completely or mor-specific transplantation antigen, sepa- partially blocked (2, 15), despite the presence of rately detected by in vivo immunological as- their corresponding mRNA's (6, 15). This obsersays. vation has led to the hypothesis that the SV40We have found that significant amounts of enhancing component functions by alleviating the two SV40-specific proteins induced by the translational block, perhaps by providing Ad2+ND2, and detected after a short pulse of ribosome specificity for mRNA recognition (22, 35S-labeled methionine, are localized on the 23), or, alternatively, by blocking the activity of plasma membrane (65% of the 56K protein and a ribosome-associated and mRNA-specific 35% of the 42K protein). This distribution did RNase (15). Our findings that a significant fracnot seem to change significantly either when tion of each of the two SV40-specific Ad2+ND2the labeling time was extended to 2 h, or when induced proteins is found tightly associated the short pulse was subsequently chased with with 40S ribosomal subunits, and that the 56K unlabeled methionine for 60 min (data not and 42K proteins found free in the cytoplasm shown). Our finding suggests that, in this loca- can bind preferentially and almost quantitation, at least one of these proteins may be the tively to 40S subunits in vitro, lend support to tumor-specific transplantation antigen, which the proposal that the SV40-specific, Ad2+ND2by definition is a cell-surface antigen (29). induced proteins may indeed function directly Moreover, we have demonstrated in other stud- as translational control elements in cells. We ies that mice preimmunized with these believe that the use of purified Ad2+ND2 proAd2+ND2-induced proteins do not support tu- teins in a well-characterized cell-free proteinmor formation upon subsequent challenge with synthesizing system will lead to better underSV40 tumor cells (G. Jay et al., unpublished standing of the molecular basis for SV40 endata). hancement, and, hence, the function of the reTogether with the earlier finding, by immu- sponsible SV40 antigen. nofluorescent staining, that U antigen has a ACKNOWLEDGMENTS diffuse cytoplasmic distribution in Ad2+ND2We thank Barrie J. Carter and Heiner Westphal for their from the nucleus infected cells and is absent (19), our observation that both of the SV40- valuable comments on the manuscript. specific Ad2+ND2 proteins are found in the cytoLITERATURE CITED plasm and not in the nucleus, even after contin- 1. Anderson, C. W., S. G. Baum, and R. F. Gesteland. uous labeling for 2 h or when the short radioac1973. Processing of adenovirus 2-induced proteins. J. Virol. 12:241-252. tive pulse was subsequently chased for 1 h
protein was found associated with the 40S ribosomes, with none located at the top of the gradient where unbound proteins have been shown to remain in the absence of added 40S ribosomes. Because of the presence of other proteins that migrated in the vicinity of the 56K protein (Fig. 5a and b), it was difficult to determine the extent of binding of the 56K protein, except to conclude that no less than 60% was associated with the 40S subunits.
VOL. 23, 1977
SV40-SPECIFIC RIBOSOME-BINDING PROTEINS
2. Baum, S. G., M. S. Horwitz, and J. V. Maizel, Jr. 1972. Studies of the mechanism of enhancement of human adenovirus infection in monkey cells by simian virus 40. J. Virol. 10:211-219. 3. Black, P. H., W. P. Rowe, H. C. Turner, and R. J. Huebner. 1963. A specific complement-fixing antigen present in SV40 tumor and transformed cells. Proc. Natl. Acad. Sci. U.S.A. 50:1148-1156. 4. Brunette, D. M., and J. E. Till. 1971. A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membr. Biol. 5:215-224. 5. Deppert, W., and G. Walter. 1976. Simian virus 40 (SV40) tumor-specific proteins in nucleus and plasma membrane of HeLa cells infected by adenovirus 2SV40 hybrid virus Ad2+ND2. Proc. Natl. Acad. Sci. U.S.A. 73:2505-2509. 6. Fox, R. I., and S. G. Baum. 1972. Synthesis of viral ribonucleic acid during restricted adenovirus infection. J. Virol. 10:220-227. 7. Freienstein, C., and G. Blobel. 1975. Nonribosomal proteins associated with eukaryotic native small ribosomal subunits. Proc. Natl. Acad. Sci. U.S.A. 72:3392-3396. 8. Girardi, A. J., and V. Defendi. 1970. Induction of SV40 transplantation antigen (TrAg) during the lytic cycle. Virology 42:688-698. 9. Grodzicker, T., C. Anderson, P. A. Sharp, and J. Sambrook. 1974. Conditional lethal mutants of adenovirus 2-simian virus 40 hybrids. I. Host range mutants of Ad2+ND,. J. Virol. 13:1237-1244. 10. Horwitz, M. S., M. D. Scharff, and J. V. Maizel, Jr. 1969. Synthesis and assembly of adenovirus 2. I. Polypeptide synthesis, assembly of capsomeres, and morphogenesis of the virion. Virology 39:682-694. 11. Jerkofsky, M. 1975. Enhancement of the replication of human adenovirus in simian cells by a series of temperature-sensitive mutants of simian virus 40. Virology 65:579-582. 12. Khera, K. S., A. Ashkenazi, F. Rapp, and J. L. Melnick. 1963. Immunity in hamsters to cells transformed in vitro and in vivo by SV40. J. Immunol. 91:604-613. 13. Kimura, G. 1974. Genetic evidence for SV40 gene function in enhancement of replication of human adenovirus in simian cells. Nature (London) 248:590-592. 14. Kit, S. 1968. Viral-induced enzymes and the problem of viral oncogenesis. Adv. Cancer Res. 11:73-221. 15. Klessig, D. F., and C. W. Anderson. 1975. Block to multiplication of adenovirus serotype 2 in monkey cells. J. Virol. 16:1650-1668. 16. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 17. Laskey, R. A., and A. D. Mills. 1975. Quantitative film detection of 3H and '4C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56:335-341.
699
18. Levine, A. S., M. J. Levin, M. N. Oxman, and A. M. Lewis, Jr. 1973. Studies of nondefective adenovirus 2simian virus 40 hybrid viruses. VII. Characterization of the simian virus 40 RNA species induced by five nondefective hybrid viruses. J. Virol. 11:672-681. 19. Lewis, A. M., Jr., A. S. Levine, C. S. Crumpacker, M. J. Levin, R. J. Samaha, and P. H. Henry. 1973. Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. V. Isolation of additional hybrids which differ in their simian virus 40-specific biological properties. J. Virol. 11:655-664. 20. Lewis, A. M., Jr., and W. P. Rowe. 1971. Studies on nondefective adenovirus-simian virus 40 hybrid viruses. I. A newly characterized simian virus 40 antigen induced by the Ad2+ND, virus. J. Virol. 7:189197. 21. Morrow, J. F., P. Berg, T. J. Kelly, Jr., and A. M. Lewis, Jr. 1973. Mapping of simian virus 40 early functions on the viral chromosome. J. Virol. 12:653658. 22. Nakajima, K., H. Ishitsuka, and K. Oda. 1974. An SV40 induced initiation factor for protein synthesis concerned with the regulation of permissiveness. Nature (London) 252:649-653. 23. Nakajima, K., and K. Oda. 1975. The alteration of ribosomes for mRNA selection concerned with adenovirus growth in SV40-infected simian cells. Virology 67:85-93. 24. O'Farrell, P. Z., L. M. Gold, and W. H. Huang. 1973. The identification of prereplicative bacteriophage T4 proteins. J. Biol. Chem. 248:5499-5501. 25. Pettersson, U., and J. Sambrook. 1973. Amount of viral DNA in the genome of cells transformed by adenovirus type 2. J. Mol. Biol. 73:125-130. 26. Rabson, A. S., G. T. O'Conor, I. K. Berezesky, and F. J. Paul. 1964. Enhancement of adenovirus growth in African green monkey kidney cell cultures by SV40. Proc. Soc. Exp. Biol. Med. 116:187-190. 27. Rapp, F., T. Kitahara, J. S. Butel, and J. L. Melnick. 1964. Synthesis of SV40 tumor antigen during replication of simian papovavirus (SV40). Proc. Natl. Acad. Sci. U.S.A. 52:1138-1142. 28. Russell, W. C., and J. J. Skehel. 1972. The polypeptides of adenovirus-infected cells. J. Gen. Virol. 15:45-57. 29. Tevethia, S. S., M. Katz, and F. Rapp. 1965. New surface antigen in cells transformed by simian papovavirus SV40. Proc. Soc. Exp. Biol. Med. 119:896-901. 30. Van der Vliet, P. C., and A. J. Levine. 1973. DNAbinding proteins specific for cells infected by adenovirus. Nature (London) New Biol. 246:170-174. 31. Velicer, L., and H. S. Ginsberg. 1970. Synthesis, transport, and morphogenesis of type 5 adenovirus capsid proteins. J. Virol. 5:338-352. 32. Walter, G., and H. Martin. 1975. Simian virus 40-specific proteins in HeLa cells infected with nondefective adenovirus 2-simian virus 40 hybrid viruses. J. Virol. 16:1236-1247.
