JOURNAL OF VIROLOGY, JUIY 1975, P. 116-122 Copyright 0 1975 American Society for Microbiology
Vol. 16, No. 1 Printed in U.S.A.
Polyoma Virus Strain with Enhanced Synthesis of Capsid Protein THOMAS G. TACHOVSKY' AND J. D. HARE*
Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication 20 February 1975
A study of the immunochemical characteristics and the synthesis of the capsid proteins of two polyoma virus strains (3049 and 1pS) was carried out to determine the mechanism responsible for the unique accumulation of those structural polypeptides in the cytoplasm of cells infected with the 3049 strain. Antisera prepared against disaggregated virus peptides and whole virus were used to measure the quantity of virus-specific antigens in cells infected by the two strains by using an indirect radioimmunoassay technique. The 3049-infected mouse embryo cells were found to contain several-fold more antibody-binding material than those infected with the lpS strain. Furthermore, the cytoplasmic fraction of 3049-infected cells also contained more antibody-binding activity, supporting the hypothesis that the phenotype of the 3049 virus (cytoplasmic capsid protein) was a reflection of the increased synthesis of the capsid polypeptides.
Viruses are known to cause a number of changes in the metabolism of host cells. Currently, investigations are concerned with the identification of virus-specific functions and the elucidation of the molecular basis of the viruscell interaction. Polyoma (Py) virus, because of its limited genetic material, is a useful tool for probing virus-cell relationships. Toward this end a series of conditional-lethal mutants has been described (4, 9). In addition to chemically induced mutant strains, several naturally occurring strains have been described (12, 15), with differences in plaque size (7, 8) or in the induction of virus-specific transplant immunity
(14).
A new variation among Py virus strains has been studied in our laboratory (6, 15). Permissive mouse cells infected with 3049 strain display both nuclear and cytoplasmic fluorescence when stained for capsid antigen by the indirect immunofluorescence technique. All other Py strains display only nuclear fluorescence (5, 17, 19, 23, 24). However, in the nonpermissive hamster embryo cell, the 3049 strain produces only nuclear fluorescence (15). This new variant is therefore unique among Py viruses in the accumulation of demonstable amounts of capsid antigen in the cytoplasm of permissive cells. Studies were undertaken to measure the quantity of virus-specific proteins synthesized by the ' Present address: Wistar Institute of Anatomy and Biology, Philadelphia, Pa. 19104.
new variant to determine whether the cytoplasmic fluorescence results from increased quantities of capsid polypeptide and possibly to elucidate the mechanism responsible for the expression of this phenotype. MATERIALS AND METHODS Virus strains. The preparation and origin of the 3049 (12) and lpS large plaque (8, 20) strains and the hemagglutination and plaque assays (11) were described previously. The stock virus used was produced in baby mouse kidney cultures (26). The rate of attachment and subsequent infection of mouse embryo cells was identical for the 3049 and lpS virus strains. Virus purification. Virus used for antiserum production and as antigen for immunological studies was purified according to the method of Roblin et al. (21). This entailed low-speed centrifugation of crude virus suspension with subsequent release of cell-bound virus by treatment of the cell debris with receptor destroying enzyme followed by fluorocarbon extraction (Genetron 113-Allied Chemical, Morristown, N.J.). Supernatant virus plus virus freed from cellular components were subjected to velocity centrifugation in KBr followed by isopycnic centrifugation in CsCl. Purified virus was concentrated either by pelleting at 100,000 x g for 2 h or by lyophilization. Virus disaggregation. Purified virus was dialyzed against distilled water and lyophilized. The resulting powder was dissolved in a solution of 8 M urea in 1 M Tris solution, pH 8.5, with 10-' M EDTA. The solution was degassed under vacuum and bubbled with nitrogen for 30 min. Two-mercaptoethanol was added to a final concentration of 0.75 M, and the
116
POLYOMA CAPSID PROTEIN SYNTHESIS
VOL. 16, 1975
117
cover slips were fixed and reacted with antiviral antibody as described for the immunofluorescence test. The cover slips were incubated for 30 min at room temperature with 125I-labeled goat anti-rabbit gamma globulin, rinsed three times for 10 min in the light-catalyzed formation of I, and a pH of 8.0 was Tris-buffered saline and counted in a gamma specmaintained by adding 2.2 M Tris base, pH 10.5. trometer. Radioimmunoassay of cellular fractions. MonoBetween pH readings the solution was maintained under nitrogen. After 2 h the S-carboxymethylated layers of mouse embryo cells were mock infected with Tris-buffered saline or infected with 3049 or lpS at a peptides were dialyzed against 0.15 M NaCl. Cell culture techniques. Mouse embryo cell cul- multiplicity of 40 PFU/cell. At 24 h postinfection the tures were prepared as described previously (13). cell monolayers were removed by trypsinization, cenHamster and rat embryo cell cultures were prepared trifuged at 800 x g for 10 min, and resuspended at 106 from 12- and 15-day pregnant animals, respectively, cells/ml in homogenization buffer-0.5 M Tris, pH by using the same method. The 3T3-Py6 cell line was 7.6, 0.15 M KCI, 3 x 10-3 M MgCl, 0.5% Nonidet, received from Thomas Benjamin (4); P388D cell line 10-3 M dithiothreitol. The cell suspension was homogfrom Allan Rabson; and Hy2a and Hy7a, mouse-ham- enized in a tight-fitting Dounce homogenizer and the ster somatic cell hybrid lines, from C. Basilico (3). extent of cell disruption was followed by phase Cells were cultivated in modified Eagle minimal microscopy. More than 90% of the cells were disrupted essential medium (AutoPow) with penicillin and after 25 strokes. The nuclei were removed by centrifustreptomycin and supplemented with either 5% or gation at 1,500 x g for 20 min and the supernatant 10% heat-inactivated bovine fetal serum. Cells for (cytoplasmic fraction) was saved. The pellet was immunofluorescence were grown in chamber slides rinsed twice with homogenizing buffer less detergent. (Lab Tek Products). All other cell culture was in Both fractions were then frozen and thawed three without glass times, sonicated for 15 s, and centrifuged at 1,500 x g plastic petri dishes (Falcon) with for 15 min before use. Supernatant fluids served as the cover slips. Production of antisera. Antisera to whole virus source of viral or control antigens. Freshly prepared extracts proved to be better were prepared and absorbed as described previously (12, 15). Antisera to disaggregated virus polypeptides sources of antigen than materials which had been stored frozen. The loss of antigenically active material were prepared by homogenizing reduced, S-carboxymethylated virus peptides in incomplete Freund with time was also observed in the immunofluoresadjuvant and inoculating 0.1 ml, containing 12 to 15 cence test. Radioimmunoassay was performed as described by ;sg of protein, into the footpads of rabbits. An additional 0.4 ml of the same protein solution in normal Hayashi et al. (16). Twenty-microliter aliquots of the saline was injected intraperitoneally. Each rabbit samples were dried in wells of polyvinyl microtiter received a similar series of injections without adju- dishes (Cooke Engineering Co., Alexandria, Va.) and vant at 2-week intervals for 6 weeks. A second fixed for 5 min with methanol. Nonimmune and antiserum to disaggregated virus polypeptides antiviral sera (0.02 ml/well) were added and the (RIPCD) was a gift of P. S. Lombardi (P. S. Lom- plates were incubated for 1 h at 37 C. After rinsing bardi, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, with distilled water 10 times, 0.02 ml of 1261-IgG fracV10, p. 196). tion of goat anti-rabbit gamma globulin (about 100,000 Fluorescent antibody technique. Fluorescent counts/min) in Tris buffer with 1% BSA was added to antibody staining was carried out as previously de- each well and incubation continued for an additional scribed (6). Fluorescein-labeled immunoglobulin G 1 h at 37 C. The plates were then rinsed 10 times with (IgG) fraction of goat anti-rabbit gamma globulin was distilled water, and the wells were separated and obtained commercially (Cappel Laboratories, Down- counted in a Packard gamma spectrometer. ington, Pa.). Immunodiffusion. Double diffusion was carried RESULTS out in 1% Noble agar containing 0.5 M glycine, 0.15 M Characteristics of antisera produced NaCl, and 0.01% merthiolate. Whole virus was applied to the plates 36 h before the application of against disaggregated virus polypeptides. capsid polypeptide preparations. The addition of up The detection of capsid antigen in the cytoto 3 M urea to the agar had no effect on the plasm of 3049-infected mouse embryo cells in development of precipitin lines. The plates were the absence of detectable numbers of virus allowed to develop for at least 48 h at 37 C in a particles (6) suggested the accumulation of a humidified chamber, washed thoroughly, dried and precursor form of the antigen. To investigate stained with 0.1% Buffalo black in 50% methanol and this possibility, antisera were prepared against 10% acetic acid solution; excess stain was removed in the reduced, S-carboxymethylated polypep2% acetic acid. 12II-labeling. The IgG fraction of goat anti-rabbit tides of both the 3049 and lpS virus strains. Care was taken to reduce the amount of crossgamma globulin was labeled with Na121 (Amersham/ reactivity of the antisera by initially injecting Searle) by the iodine monochloride technique (2). Indirect radioimmunoassay. Cells grown on glass small amounts of antigen emulsified in incomwas allowed to stand for 2 to 4 h at room temperature. An aliquot of freshly prepared solution of 1.2 M iodoacetamide in 1 M Tris, pH 8.5, was added to a final concentration of 0.37 M iodoacetamide. The tube was covered with tin foil to prevent
solution
or
118
TACHOVSKY AND HARE
J. VIROL.
plete Freund adjuvant (25). Table 1 lists the characteristics of the antisera produced in rabbits against intact 3049 virions (anti-3049 pool) as well as 3049 (anti3049D) and lpS (anti-1pSD) polypeptides. Neither of the antisera to disaggregated polypeptides possessed neutralizing or hemagglutination-inhibiting activity against the intact virus. Both sera, however, reacted with the viral antigens in the infected cells detected by immunofluorescence, as shown in Table 2. The distribution of fluorescence was found to be identical to that produced when the whole virus antiserum was used. The immunological reactivities of these antisera were examined further by immunodiffusion. As shown diagrammatically in Fig. 1A, anti-3049 pool serum reacted with both 3049 and lpS virus particles to form a heavy precipitin line that showed a reaction of identity. This whole virus antiserum, however, did not precipitate with disaggregated virus proteins. A small spur frequently formed in the reaction between 3049 antiserum and the 3049 antigen (Fig. 1A), demonstrating that there was, in addition to the major shared determinant, a second antigenic determinant present on the 3049 virion not represented on the lpS virion. By contrast, precipitin lines were formed in the reaction between antisera to disaggregated virus proteins and the virus polypeptide, but not with intact virions. These reactions are shown in Fig. 1B in which anti-3049D serum is included; anti-lpSD serum showed an identical pattern. The precipitin lines between the disagTABLE 1. Some characteristics of antisera against whole and disaggregated virus Antibody titera Test
Anti-3049
pool
Hemagglutination inhibitionb Plaque neutraliza-
Anti- | 3049D
Anti-1S
D
1,260
40c
40c
400,000
loc
loc
640
160'
80'
tiond
Immunofluorescencee
aReciprocal of the last dilution positive for each test. "Dilution of serum sufficient to inhibit 8 hemagglutinating units of both SE 3049 and lpS strains. cPreimmunization serum displayed similar activity. d Dilution of serum necessary to produce 50% inhibition of plaque formation by both virus strains. t Preimmunization serum displayed no reaction with infected cells.
TABLE 2. Distribution of fluorescence at 24 h postinfection in mouse embryo cells infected with SE 3049 and IpS viruses lpSb
3049b
Antiseruma
Anti-3049 pool Anti-210 Rl-PCDd Anti-3049De Anti 1pSDe
Nucleus
Cytoplasm
+c +
+ +
+
+
+
+
+
+
+
_
+
+
+
_
Nucleus
Cytoplasm
+
c
_
aAll sera used at 1:10 dilution in complete Tris buffer. b Multiplicity of infection, 40 PFU/cell. c +, Fluorescence; -, no fluorescence. dAntiserum prepared against disaggregated polyoma and received from P. Lombardi. eAntisera against disaggregated polyoma prepared for this study.
A
B e
FIG. 1. Double diffusion gel patterns. Symbols: A, anti-3049 pool antiserum; B, anti-3049 polypeptide antiserum. A3, anti-3049 pool; A3D, anti-3049 polypeptide; 3W, 3049 virion; Sw, IpS virion; 3D, 3049 polypeptide; SD, IpS polypeptide.
gregated polypeptides of the two virus strains and their respective antisera showed reactions of identity with no evidence for the formation of a similar spur as seen in the whole virus system. These reactions demonstrate that the intact virions of both virus strains carry one major antigenic determinant that is common in the two strains but which is distinct from the major determinant on the dissociated, S-carboxymethylated polypeptides of the two strains. In spite of these differences, antisera to intact virus and to the polypeptides detect their respective antigenic determinants in the cytoplasm and nucleus of the 3049-infected cells but only in the nucleus of lpS-infected cells (Table 2). Expression of the 3049 phenotype in various host cells. Previous studies (6, 15) have shown that hamster embryo cells infected with the 3049 strain of Py display only nuclear
POLYOMA CAPSID PROTEIN SYNTHESIS
VOL. 16, 1975
fluorescence in the small proportion of cells in which lytic infection takes place. The failure of hamster embryo cells to accumulate capsid antigen in the cytoplasm suggested a unique virus-cell interaction in which the synthesis of capsid polypeptide was regulated by the host cell. A variety of cells were then investigated for their effect on the expression of this unique phenotype and the results are summarized in Table 3. Cells of mouse origin, including both primary cell cultures and established cell lines, all displayed cytoplasmic fluorescence when tested with anti-3049 pool serum for capsid antigens 24 h after infection. Of special interest was the expression of the 3049 phenotypic character in the 3T3-Py6 cells, a cell line established by the transformation of 3T3 mouse cells by a host range mutant of Py (4). The presumed presence of another Py genome had no effect on the 3049 phenotype. Mouse-hamster somatic cell hybrid lines Hy7a and Hy2a (3) were also examined. Both virus strains replicated in each line (unpublished data); the same fluorescent patterns were observed in these cell lines as in permissive mouse embryo cells. The presence of hamster chromosomes in these two cell lines did not suppress the cytoplasmic fluorescence phenotype. Two other cell types were also tested. Primary baby hamster kidney cells displayed 0.1 to 0.2%
positive cells in the immunofluorescence test. The distribution of fluorescence in these cells was identical to that observed in hamster embryo cells. On the other hand, rat embryo fibroblasts not only displayed a greater percentage of cells (15 to 20%) producing capsid polypeptide but the cells infected with 3049 virus were positive in both nuicleus and cytoplasm. Measurement of capsid proteins by quantitative radioimmunoassay. To measure the relative quantity of capsid proteins or polypeptides produced in mouse cells infected with the 3049 or lpS virus strains, the indirect radioimmunoassay technique described by Hayashi et al. (16) was utilized. The binding of 125I-labeled goat anti-rabbit gamma globulin to cells treated with rabbit anti-3049 pool serum at different times after infection is shown in Fig. 2. Capsid antigen was detected in equivalent amounts in cells infected with both viruses at 20 h. From that point on, however, 3049-infected cells bound increasingly greater quantities of the labeled antiserum than lpS-infected cells. This increased binding of 12I-antiglobulin was not the result of a greater proportion of cells becoming infected by the 3049 virus as indicated by the similar number of cells synthesizing capsid proteins in the two infections, as measured by immunofluorescence, but rather reflected a 10-
TABLE 3. Capsid polypeptide distribution in various cell types as determined by indirect immunofluorescence 3049
Cell typea
Nucleus
(43S)
8-
4{0%)
lpS
Cyto-
Nucleus
plasm
6-
Cyto-
plasm 1)c
Mouse MEF P 388 D1 BMK 3T3-Py6 Mouse-hamster hybrid Hy7a
+b
119
(19%)
4-
+ + +
+ + + +
+ + + +
+ +
+ +
+ +
-
Hy2a Hamster BHK
+
+
-
(40)
-
-
-
-
-
2-
(154)
0 ,. 0
.
10
.
. . 20
)-(36X)
)( 28S 1
30
1
TIME POST-INFECTION (Hours)
FIG. 2. Binding of 125I-antiglobulin to infected mouse enbryo cells treated with anti-3049 pool antiREF + + + serum in the indirect radioimmunoassay. Numbers in a Cells were grown in chamber slides and fixed and parenthesis indicate percentage of cells positive for stained at 24 h postinfection, except baby hamster capsid antigen as determined by the indirect immukidney and rat embryo fibroblasts, which were fixed at nofluorescence test. Each point represents the average 48 h postinfection. of five determinations. Symbols: (A) SE3049; (U) b+, Fluorescence; -, fluorescence negative. ips.
Rat
120
J. VIROL.
TACHOVSKY AND HARE
greater amount of antigenic material in 3049infected cells. Radioimmunoassay of capsid proteins in subcellular fractions. The demonstration of more antibody-binding material in 3049infected cells supported the hypothesis that more capsid polypeptide was synthesized. An attempt was made to demonstrate that more polypeptide was present in the cytoplasmic fraction of infected cells. Table 4 shows the results obtained by indirect radioimmunoassay using the nuclear and cytoplasmic fractions of virus-infected cells. In each case 3049-infected cell fractions, both nuclear and cytoplasmic, bound more '25I-antiglobulin than the lpS fractions. Significantly more antigen was detectable in the cytoplasmic fraction of the 3049-infected cells by the anti-disaggregated virus antisera than by the anti-whole virus antiserum. The nuclear fractions of both infected cell populations contained detectable viral antigen, the reacting species present in a form recognizable by both the anti-whole virus antiserum and the anti-disaggregated virus antisera. Inhibition of capsid protein synthesis by metabolic inhibitors. The effect of several metabolic inhibitors on the synthesis of capsid proteins as measured by the indirect radioimmunoassay in 3049 virus-infected cells was then studied. As shown in Fig. 3, fluorodeoxyuridine, actinomycin D, a-amanitin and cycloheximide all strongly inhibited synthesis of the antibodybinding protein when added 1 h after infection, followed by a progressive reduction in the degree of inhibition during the course of infection. Several interesting points should be noted. The synthesis of capsid antigen was as dependent on continuing DNA synthesis as it was on RNA synthesis. The effect of ca-amanitin, an inhibitor
*FdU
800
ACYCLOHEXIMIDE
~605040-
05 3020-
O-'4,.......
100
5
10
15
20
25
30
TIME OF ADDITION OF INHIBITOR (Hrs post-infection) FIG. 3. The effect of various metabolic inhibitors on the synthesis of capsid proteins in 3049-infected mouse embryo cells as measured by binding of 125J_ antiglobulin to cells treated with anti-3049 pool antiserum. The inhibitors were added at different times after infection and all cover slips were harvested at 30 h postinfection. Results are expressed as a percentage of binding to control, untreated cells at 30 h and are charted at the time when the inhibitor was added. Actinomycin D, 0.05 ,ug/ml; a-amanitin, 1 Asg/ml; fluorodeoxyuridine (FdU), 15 A.g/ml; cycloheximide, 1 lAg/ ml. Five duplicate cover slip samples were assayed for each determination.
of nucleoplasmic RNA polymerase II (18), was lost more rapidly than that of actinomycin D, in spite of the fact that it was fully as active as actinomycin D when added early in infection. Finally, as would be expected, cycloheximide inhibited synthesis of the protein for a longer time than any of the other compounds.
DISCUSSION
The 3049 strain of Py virus displays a uniquely different phenotype from other reported strains in that capsid polypeptide is TABLE 4. Subcellular distribution of capsid detectable in the cytoplasm as well as the polypeptide in IpS- and 3049-infected mouse embryo nucleus of the infected mouse cell. Several radioimmunoassay cells as determined by indirect possible mechanisms for this phenotype were considered, namely, altered diffusion of capsid Counts/min per welIb protein from cytoplasm to nucleus, or enhanced Anti-3049 Anti-3049D Anti l SD synthesis of capsid protein (6). This study was Antigena carried out to measure the relative amounts of capsid polypeptide in the whole infected cell or 210 + 9 210 + 8 SE 3049/cytoplasm 60 ± 2 0 0 40 ± 2 fractions thereof, following infection with the lpS/cytoplasm SE 3049/nucleus 550 ± 15 730 ± 16 400 + 6 3049 virus and a control, wild-type virus (1pS) 280 ± 11 320 ± 9 170 4 3 lpS/nucleus using more quantitative, radioimmunoassay techniques. aCells were cultured in 100-mm petri dishes inAntisera to intact virions as well as the fected with virus at a multiplicity of 40 PFU/cell, and dissociated, S-carboxymethylated polypeptides harvested 24 h later. bMean ±e 1 standard deviation for three determina- derived from purified virions were prepared and characterized by several different criteria. Antitions.
VOL. 16, 1975
serum to intact 3049 virions detected a major common antigenic determinant on the 3049 and lpS particle but did not react with the dissociated polypeptides. Antisera to the disaggregated, S-carboxymethylated polypeptides of either 3049 or lpS virions detected a major common antigenic determinant on the dissociated polypeptides of both virus strains. These sera did not react with the intact virion of either strain as measured by neutralization of infectivity, inhibition of hemagglutination, or formation of precipitin bands. These data indicated that the capsid polypeptides of the two virus strains were immunochemically indistinguishable and therefore the use of a radioimmunoassay to measure the relative quantities of capsid proteins in the cells infected with the two virus strains was valid. Furthermore, these reagents could be utilized to identify precursor polypeptides as well as the protein configuration characteristic of intact virions. With these reagents it was then shown that the cytoplasm as well as the nucleus of the 3049-infected cell contained virus-specific polypeptides that reacted with antisera to both intact virions and the dissociated polypeptides. The lpS-infected cell, however, showed evidence for the presence of polypeptides carrying the two antigenic determinants only in the nucleus. These findings support the concept that the capsid polypeptide is synthesized in the cytoplasm and is rapidly translocated to the nucleus in the wild-type infection where particle assembly takes place. The precursor molecule, however, never attains a level sufficient to be observed in the cytoplasm with the indirect fluorescent antibody technique. In the case of the 3049 strain, infection is associated with the production of the polypeptide resulting in a concentration in the cytoplasm sufficient to react positively with the fluorescent antibody technique. Furthermore, the demonstration that the antigenic determinants characteristic of both intact virions and precursor polypeptides are found in the cytoplasm as well as the nucleus suggests that some structural maturation may also take place in the cytoplasm. The next step was to measure the relative quantity of virus-specfic antigenic material in cells infected with 3049 and lpS viruses using an indirect radioimmunoassay as described by Hayashi et al. (16). By using carefully controlled infectious doses of each virus strain to yield equal numbers of cells producing capsid protein detected by immunofluorescence, it was possible to demonstrate that 3049-infected cells bind several-fold more virus-specific antiserum than lpS-infected cells. This was detected by
POLYOMA CAPSID PROTEIN SYNTHESIS
121
the specific binding of 125I-labeled IgG fraction of goat anti-rabbit gamma globulin. Furthermore, the cytoplasmic fraction of the 3049infected cell was shown to contain antibodybinding antigen under conditions in which the cytoplasm of lpS-infected cells failed to bind antibody. These findings provide direct evidence that the synthesis of capsid protein is significantly higher in 3049- than in lpS-infected cells and that there is a significant accumulation of capsid polypeptide precursor molecules in the cytoplasm of the 3049-infected cell. Recently it has been demonstrated that there is also a twofold greater quantity of virus-specific polyadenylated RNA in both the nuclear RNA and polyribosomal fractions of 3049-infected cells as compared to those infected with lpS virus (22). On this basis, then, it can be proposed that the phenotype of the 3049 virus, namely, the presence of capsid polypeptide molecules in the cytoplasm, is the direct result of the increased numbers of mRNA templates specifying at least the major structural protein, VP2 (21), rather than some virus controlled enhancement of the translation rate from the mRNA. Direct -assessment of the rate of translation or the quantity of translatable mRNA has not been carried out, however. Two other findings bear on the question as to which level of genetic control is altered in the 3049 virus replication process. The first is the demonstration that the expression of the phenotype is closely correlated with the presence of certain mouse chromosomes such that in the absence of mouse genetic information, i.e., in hamster cells, the phenotype is suppressed. On the other hand, in mouse-hamster hybrids with sufficient mouse DNA represented, the virus replicates and the phenotype is expressed. The rat embryo cell is of interest in that a significant number of cells are permissive for the expression of "late" functions and the 3049 phenotype is expressed. These findings suggest that some host cell function is intimately associated with the expression of the phenotype. A second finding of interest is the prolonged sensitivity of the synthesis of capsid protein to inhibition of DNA synthesis by FdU, with a pattern quite similar to that produced by actinomycin D (Fig. 3). This is reminiscent of the demonstration by Glover (10) that inhibition of Py DNA synthesis restricts the quantity and quality of the viral RNA transcribed "late" in infection to yield an RNA population that resembles "early" transcripts. Taken together, these findings suggest that the rate of synthesis of this late virus gene
122
TACHOVSKY AND HARE
product, the major capsid protein, is regulated by the quantity of mRNA available for translation. Furthermore, the quantity of mRNA may in turn be controlled by some host cell function which determines the rate of transcription of the viral genome following the onset of viral DNA synthesis. This function could be either a host DNA or RNA polymerase the function of which is modified by some factor associated with the synthesis of late viral functions. The reverse transcription co-factor postulated by Amati (1) would also fit with this postulate. The elucidation of the mechanism of this unique phenotype should provide a significant increase in the understanding of genetic regulation in eukaryotic host cell-virus systems. LITERATURE CITED 1. Amati, P. 1974. A working model for oncogenic DNA virus replication. J. Theor. Biol. 46:221-227. 2. Bale, W. F., R. W. Helmkamp, T. P. Davis, M. J. Izzo, R. L. Goodland, M. A. Contreras, and I. L. Spar. 1966. High specific activity labeling of protein with 131I by the iodine monochloride method. Proc. Soc. Exp. Biol. Med. 122:407-414. 3. Basilico, C., Y. Matsuya, and H. Green. 1970. The interaction of polyoma virus with mouse-hamster hybrid cells. Virology 41:295-305. 4. Benjamin, T. L. 1970. Host range mutants of polyoma virus. Proc. Natl. Acad. Sci. U.S.A. 67:390-399. 5. Bereczky, E., R. Hughes, J. M. Bowen, W. Munyon, and L. Dmochowski. 1965. Study of DNA synthesis and antigen formation in polyoma virus-infected mouse embryo cells by autoradiography and immunofluorescence. Texas Rep. Biol. Med. 23:3-15. 6. Betts, R. F., T. G. Tachovsky, and J. D. Hare. 1972. Studies on the mechanism of cytoplasmic antigen accumulation following infection with a new variant of polyoma virus. J. Gen. Virol. 16:29-38. 7. Crawford, L. V. 1962. The adsorption of polyoma virus. Virology 18:177-181. 8. Diamond, L., and L. V. Crawford. 1964. Some characteristics of large-plaque and small-plaque lines of polyoma virus. Virology 22:235-244. 9. Eckhart, W. 1969. Complementation and transformation by temperature-sensitive mutants of polyoma virus. Virology 38:120-125. 10. Glover, D. M. 1974. Coupling of polyoma DNA and RNA synthesis. Biochem. Biophys. Res. Commun. 57:1137-1143. 11. Hare, J. D., P. Balduzzi, and H. R. Morgan. 1963.
J. VIROL.
12. 13.
14.
15. 16. 17.
18.
19.
20.
21. 22.
23. 24.
25. 26.
Polyoma virus and L cell relationship. I. Some characteristics of a cell line persistently infected with polyoma virus. J. Natl. Cancer Inst. 30:45-56. Hare, J. D., and J. Chan. 1966. Antigenic variation among polyoma viruses from different sources demonstrated by plaque neutralization. Virology 30:62-73. Hare, J. D. 1967. Localization and characterization of the phenylalanine transport mechanism in normal and polyoma-transformed hamster cells. Cancer Res. 27:2357-2363. Hare, J. D. 1967. Transplant immunity to polyomainduced tumor cells. IV. A polyoma strain defective in transplant antigen induction. Virology 31:625-632. Hare, J. D. 1970. A new type of variation among polyoma virus characterized by cytoplasmic accumulation of capsid antigen. Virology 40:978-988. Hayashi, K., J. Rosenthal. and A. L. Notkins. 1972. Iodine-125 labeled antibody to viral antigens binding to the surface of virus-infected cells. Science 176:516-518. Henle, G., F. Deinhardt, and J. Rodrigues. 1959. The development of polyoma virus in mouse embryo cells as revealed by fluorescent antibody staining. Virology 8:388-391. Lindell, T. J., F. Weinberg, P. W. Morris, R. G. Roeder, and W. J. Rutter. 1970. Specific inhibition of nuclear RNA polymerase II by a-amanitin. Science 170:447-449. Malmgren, R. A., R. Rabatti, and A. S. Rabson. 1960. Intracellular localization of polyoma virus antigen demonstrated with fluorescein labelled antisera. J. Natl. Cancer Inst. 24:581-588. McCulloch, E. A., A. F. Howatson, L. Siminovitch, A. A. Axelrad, and A. W. Ham. 1959. Tumor induction in Swiss mice by virus obtained originally from a mammary tumor in C3H/Jax mouse and then cultivated in vitro. Proc. Am. Assoc. Cancer Res. 3:41. Roblin, R., E. Harle, and R. Dulbecco. 1971. Polyoma virus proteins. I. Multiple virion components. Virology 45:555-566. Rutherford, R. B., and J. D. Hare. 1974. Evidence for a regulatory function related to the expression of the polyoma genome following the onset of virus DNA replication. Biochem. Biophys. Res. Commun 58:839-846. Sachs, L., and M. Fogel. 1960. Polyoma virus synthesis in tumor cells as measured by the fluorescent antibody technique. Virology 11:722-736. Williams, M. G., and R. Sheinin. 1961. Cytological studies of mouse embryo cells infected with polyoma virus using acridine orange and fluorescent antibody. Virology 13:368-369. Vestergard-Jorgensen, P. E. 1972. Freund's adjuvants: their influence on the specificity of viral antisera. Acta Pathol. Microbiol. Scand. Sect. B 80:931-933. Winocour, E. 1963. Purification of polyoma virus. Virology 19:158-168.
Vol. 16, No. 1 Printed in U.S.A.
Polyoma Virus Strain with Enhanced Synthesis of Capsid Protein THOMAS G. TACHOVSKY' AND J. D. HARE*
Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication 20 February 1975
A study of the immunochemical characteristics and the synthesis of the capsid proteins of two polyoma virus strains (3049 and 1pS) was carried out to determine the mechanism responsible for the unique accumulation of those structural polypeptides in the cytoplasm of cells infected with the 3049 strain. Antisera prepared against disaggregated virus peptides and whole virus were used to measure the quantity of virus-specific antigens in cells infected by the two strains by using an indirect radioimmunoassay technique. The 3049-infected mouse embryo cells were found to contain several-fold more antibody-binding material than those infected with the lpS strain. Furthermore, the cytoplasmic fraction of 3049-infected cells also contained more antibody-binding activity, supporting the hypothesis that the phenotype of the 3049 virus (cytoplasmic capsid protein) was a reflection of the increased synthesis of the capsid polypeptides.
Viruses are known to cause a number of changes in the metabolism of host cells. Currently, investigations are concerned with the identification of virus-specific functions and the elucidation of the molecular basis of the viruscell interaction. Polyoma (Py) virus, because of its limited genetic material, is a useful tool for probing virus-cell relationships. Toward this end a series of conditional-lethal mutants has been described (4, 9). In addition to chemically induced mutant strains, several naturally occurring strains have been described (12, 15), with differences in plaque size (7, 8) or in the induction of virus-specific transplant immunity
(14).
A new variation among Py virus strains has been studied in our laboratory (6, 15). Permissive mouse cells infected with 3049 strain display both nuclear and cytoplasmic fluorescence when stained for capsid antigen by the indirect immunofluorescence technique. All other Py strains display only nuclear fluorescence (5, 17, 19, 23, 24). However, in the nonpermissive hamster embryo cell, the 3049 strain produces only nuclear fluorescence (15). This new variant is therefore unique among Py viruses in the accumulation of demonstable amounts of capsid antigen in the cytoplasm of permissive cells. Studies were undertaken to measure the quantity of virus-specific proteins synthesized by the ' Present address: Wistar Institute of Anatomy and Biology, Philadelphia, Pa. 19104.
new variant to determine whether the cytoplasmic fluorescence results from increased quantities of capsid polypeptide and possibly to elucidate the mechanism responsible for the expression of this phenotype. MATERIALS AND METHODS Virus strains. The preparation and origin of the 3049 (12) and lpS large plaque (8, 20) strains and the hemagglutination and plaque assays (11) were described previously. The stock virus used was produced in baby mouse kidney cultures (26). The rate of attachment and subsequent infection of mouse embryo cells was identical for the 3049 and lpS virus strains. Virus purification. Virus used for antiserum production and as antigen for immunological studies was purified according to the method of Roblin et al. (21). This entailed low-speed centrifugation of crude virus suspension with subsequent release of cell-bound virus by treatment of the cell debris with receptor destroying enzyme followed by fluorocarbon extraction (Genetron 113-Allied Chemical, Morristown, N.J.). Supernatant virus plus virus freed from cellular components were subjected to velocity centrifugation in KBr followed by isopycnic centrifugation in CsCl. Purified virus was concentrated either by pelleting at 100,000 x g for 2 h or by lyophilization. Virus disaggregation. Purified virus was dialyzed against distilled water and lyophilized. The resulting powder was dissolved in a solution of 8 M urea in 1 M Tris solution, pH 8.5, with 10-' M EDTA. The solution was degassed under vacuum and bubbled with nitrogen for 30 min. Two-mercaptoethanol was added to a final concentration of 0.75 M, and the
116
POLYOMA CAPSID PROTEIN SYNTHESIS
VOL. 16, 1975
117
cover slips were fixed and reacted with antiviral antibody as described for the immunofluorescence test. The cover slips were incubated for 30 min at room temperature with 125I-labeled goat anti-rabbit gamma globulin, rinsed three times for 10 min in the light-catalyzed formation of I, and a pH of 8.0 was Tris-buffered saline and counted in a gamma specmaintained by adding 2.2 M Tris base, pH 10.5. trometer. Radioimmunoassay of cellular fractions. MonoBetween pH readings the solution was maintained under nitrogen. After 2 h the S-carboxymethylated layers of mouse embryo cells were mock infected with Tris-buffered saline or infected with 3049 or lpS at a peptides were dialyzed against 0.15 M NaCl. Cell culture techniques. Mouse embryo cell cul- multiplicity of 40 PFU/cell. At 24 h postinfection the tures were prepared as described previously (13). cell monolayers were removed by trypsinization, cenHamster and rat embryo cell cultures were prepared trifuged at 800 x g for 10 min, and resuspended at 106 from 12- and 15-day pregnant animals, respectively, cells/ml in homogenization buffer-0.5 M Tris, pH by using the same method. The 3T3-Py6 cell line was 7.6, 0.15 M KCI, 3 x 10-3 M MgCl, 0.5% Nonidet, received from Thomas Benjamin (4); P388D cell line 10-3 M dithiothreitol. The cell suspension was homogfrom Allan Rabson; and Hy2a and Hy7a, mouse-ham- enized in a tight-fitting Dounce homogenizer and the ster somatic cell hybrid lines, from C. Basilico (3). extent of cell disruption was followed by phase Cells were cultivated in modified Eagle minimal microscopy. More than 90% of the cells were disrupted essential medium (AutoPow) with penicillin and after 25 strokes. The nuclei were removed by centrifustreptomycin and supplemented with either 5% or gation at 1,500 x g for 20 min and the supernatant 10% heat-inactivated bovine fetal serum. Cells for (cytoplasmic fraction) was saved. The pellet was immunofluorescence were grown in chamber slides rinsed twice with homogenizing buffer less detergent. (Lab Tek Products). All other cell culture was in Both fractions were then frozen and thawed three without glass times, sonicated for 15 s, and centrifuged at 1,500 x g plastic petri dishes (Falcon) with for 15 min before use. Supernatant fluids served as the cover slips. Production of antisera. Antisera to whole virus source of viral or control antigens. Freshly prepared extracts proved to be better were prepared and absorbed as described previously (12, 15). Antisera to disaggregated virus polypeptides sources of antigen than materials which had been stored frozen. The loss of antigenically active material were prepared by homogenizing reduced, S-carboxymethylated virus peptides in incomplete Freund with time was also observed in the immunofluoresadjuvant and inoculating 0.1 ml, containing 12 to 15 cence test. Radioimmunoassay was performed as described by ;sg of protein, into the footpads of rabbits. An additional 0.4 ml of the same protein solution in normal Hayashi et al. (16). Twenty-microliter aliquots of the saline was injected intraperitoneally. Each rabbit samples were dried in wells of polyvinyl microtiter received a similar series of injections without adju- dishes (Cooke Engineering Co., Alexandria, Va.) and vant at 2-week intervals for 6 weeks. A second fixed for 5 min with methanol. Nonimmune and antiserum to disaggregated virus polypeptides antiviral sera (0.02 ml/well) were added and the (RIPCD) was a gift of P. S. Lombardi (P. S. Lom- plates were incubated for 1 h at 37 C. After rinsing bardi, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, with distilled water 10 times, 0.02 ml of 1261-IgG fracV10, p. 196). tion of goat anti-rabbit gamma globulin (about 100,000 Fluorescent antibody technique. Fluorescent counts/min) in Tris buffer with 1% BSA was added to antibody staining was carried out as previously de- each well and incubation continued for an additional scribed (6). Fluorescein-labeled immunoglobulin G 1 h at 37 C. The plates were then rinsed 10 times with (IgG) fraction of goat anti-rabbit gamma globulin was distilled water, and the wells were separated and obtained commercially (Cappel Laboratories, Down- counted in a Packard gamma spectrometer. ington, Pa.). Immunodiffusion. Double diffusion was carried RESULTS out in 1% Noble agar containing 0.5 M glycine, 0.15 M Characteristics of antisera produced NaCl, and 0.01% merthiolate. Whole virus was applied to the plates 36 h before the application of against disaggregated virus polypeptides. capsid polypeptide preparations. The addition of up The detection of capsid antigen in the cytoto 3 M urea to the agar had no effect on the plasm of 3049-infected mouse embryo cells in development of precipitin lines. The plates were the absence of detectable numbers of virus allowed to develop for at least 48 h at 37 C in a particles (6) suggested the accumulation of a humidified chamber, washed thoroughly, dried and precursor form of the antigen. To investigate stained with 0.1% Buffalo black in 50% methanol and this possibility, antisera were prepared against 10% acetic acid solution; excess stain was removed in the reduced, S-carboxymethylated polypep2% acetic acid. 12II-labeling. The IgG fraction of goat anti-rabbit tides of both the 3049 and lpS virus strains. Care was taken to reduce the amount of crossgamma globulin was labeled with Na121 (Amersham/ reactivity of the antisera by initially injecting Searle) by the iodine monochloride technique (2). Indirect radioimmunoassay. Cells grown on glass small amounts of antigen emulsified in incomwas allowed to stand for 2 to 4 h at room temperature. An aliquot of freshly prepared solution of 1.2 M iodoacetamide in 1 M Tris, pH 8.5, was added to a final concentration of 0.37 M iodoacetamide. The tube was covered with tin foil to prevent
solution
or
118
TACHOVSKY AND HARE
J. VIROL.
plete Freund adjuvant (25). Table 1 lists the characteristics of the antisera produced in rabbits against intact 3049 virions (anti-3049 pool) as well as 3049 (anti3049D) and lpS (anti-1pSD) polypeptides. Neither of the antisera to disaggregated polypeptides possessed neutralizing or hemagglutination-inhibiting activity against the intact virus. Both sera, however, reacted with the viral antigens in the infected cells detected by immunofluorescence, as shown in Table 2. The distribution of fluorescence was found to be identical to that produced when the whole virus antiserum was used. The immunological reactivities of these antisera were examined further by immunodiffusion. As shown diagrammatically in Fig. 1A, anti-3049 pool serum reacted with both 3049 and lpS virus particles to form a heavy precipitin line that showed a reaction of identity. This whole virus antiserum, however, did not precipitate with disaggregated virus proteins. A small spur frequently formed in the reaction between 3049 antiserum and the 3049 antigen (Fig. 1A), demonstrating that there was, in addition to the major shared determinant, a second antigenic determinant present on the 3049 virion not represented on the lpS virion. By contrast, precipitin lines were formed in the reaction between antisera to disaggregated virus proteins and the virus polypeptide, but not with intact virions. These reactions are shown in Fig. 1B in which anti-3049D serum is included; anti-lpSD serum showed an identical pattern. The precipitin lines between the disagTABLE 1. Some characteristics of antisera against whole and disaggregated virus Antibody titera Test
Anti-3049
pool
Hemagglutination inhibitionb Plaque neutraliza-
Anti- | 3049D
Anti-1S
D
1,260
40c
40c
400,000
loc
loc
640
160'
80'
tiond
Immunofluorescencee
aReciprocal of the last dilution positive for each test. "Dilution of serum sufficient to inhibit 8 hemagglutinating units of both SE 3049 and lpS strains. cPreimmunization serum displayed similar activity. d Dilution of serum necessary to produce 50% inhibition of plaque formation by both virus strains. t Preimmunization serum displayed no reaction with infected cells.
TABLE 2. Distribution of fluorescence at 24 h postinfection in mouse embryo cells infected with SE 3049 and IpS viruses lpSb
3049b
Antiseruma
Anti-3049 pool Anti-210 Rl-PCDd Anti-3049De Anti 1pSDe
Nucleus
Cytoplasm
+c +
+ +
+
+
+
+
+
+
+
_
+
+
+
_
Nucleus
Cytoplasm
+
c
_
aAll sera used at 1:10 dilution in complete Tris buffer. b Multiplicity of infection, 40 PFU/cell. c +, Fluorescence; -, no fluorescence. dAntiserum prepared against disaggregated polyoma and received from P. Lombardi. eAntisera against disaggregated polyoma prepared for this study.
A
B e
FIG. 1. Double diffusion gel patterns. Symbols: A, anti-3049 pool antiserum; B, anti-3049 polypeptide antiserum. A3, anti-3049 pool; A3D, anti-3049 polypeptide; 3W, 3049 virion; Sw, IpS virion; 3D, 3049 polypeptide; SD, IpS polypeptide.
gregated polypeptides of the two virus strains and their respective antisera showed reactions of identity with no evidence for the formation of a similar spur as seen in the whole virus system. These reactions demonstrate that the intact virions of both virus strains carry one major antigenic determinant that is common in the two strains but which is distinct from the major determinant on the dissociated, S-carboxymethylated polypeptides of the two strains. In spite of these differences, antisera to intact virus and to the polypeptides detect their respective antigenic determinants in the cytoplasm and nucleus of the 3049-infected cells but only in the nucleus of lpS-infected cells (Table 2). Expression of the 3049 phenotype in various host cells. Previous studies (6, 15) have shown that hamster embryo cells infected with the 3049 strain of Py display only nuclear
POLYOMA CAPSID PROTEIN SYNTHESIS
VOL. 16, 1975
fluorescence in the small proportion of cells in which lytic infection takes place. The failure of hamster embryo cells to accumulate capsid antigen in the cytoplasm suggested a unique virus-cell interaction in which the synthesis of capsid polypeptide was regulated by the host cell. A variety of cells were then investigated for their effect on the expression of this unique phenotype and the results are summarized in Table 3. Cells of mouse origin, including both primary cell cultures and established cell lines, all displayed cytoplasmic fluorescence when tested with anti-3049 pool serum for capsid antigens 24 h after infection. Of special interest was the expression of the 3049 phenotypic character in the 3T3-Py6 cells, a cell line established by the transformation of 3T3 mouse cells by a host range mutant of Py (4). The presumed presence of another Py genome had no effect on the 3049 phenotype. Mouse-hamster somatic cell hybrid lines Hy7a and Hy2a (3) were also examined. Both virus strains replicated in each line (unpublished data); the same fluorescent patterns were observed in these cell lines as in permissive mouse embryo cells. The presence of hamster chromosomes in these two cell lines did not suppress the cytoplasmic fluorescence phenotype. Two other cell types were also tested. Primary baby hamster kidney cells displayed 0.1 to 0.2%
positive cells in the immunofluorescence test. The distribution of fluorescence in these cells was identical to that observed in hamster embryo cells. On the other hand, rat embryo fibroblasts not only displayed a greater percentage of cells (15 to 20%) producing capsid polypeptide but the cells infected with 3049 virus were positive in both nuicleus and cytoplasm. Measurement of capsid proteins by quantitative radioimmunoassay. To measure the relative quantity of capsid proteins or polypeptides produced in mouse cells infected with the 3049 or lpS virus strains, the indirect radioimmunoassay technique described by Hayashi et al. (16) was utilized. The binding of 125I-labeled goat anti-rabbit gamma globulin to cells treated with rabbit anti-3049 pool serum at different times after infection is shown in Fig. 2. Capsid antigen was detected in equivalent amounts in cells infected with both viruses at 20 h. From that point on, however, 3049-infected cells bound increasingly greater quantities of the labeled antiserum than lpS-infected cells. This increased binding of 12I-antiglobulin was not the result of a greater proportion of cells becoming infected by the 3049 virus as indicated by the similar number of cells synthesizing capsid proteins in the two infections, as measured by immunofluorescence, but rather reflected a 10-
TABLE 3. Capsid polypeptide distribution in various cell types as determined by indirect immunofluorescence 3049
Cell typea
Nucleus
(43S)
8-
4{0%)
lpS
Cyto-
Nucleus
plasm
6-
Cyto-
plasm 1)c
Mouse MEF P 388 D1 BMK 3T3-Py6 Mouse-hamster hybrid Hy7a
+b
119
(19%)
4-
+ + +
+ + + +
+ + + +
+ +
+ +
+ +
-
Hy2a Hamster BHK
+
+
-
(40)
-
-
-
-
-
2-
(154)
0 ,. 0
.
10
.
. . 20
)-(36X)
)( 28S 1
30
1
TIME POST-INFECTION (Hours)
FIG. 2. Binding of 125I-antiglobulin to infected mouse enbryo cells treated with anti-3049 pool antiREF + + + serum in the indirect radioimmunoassay. Numbers in a Cells were grown in chamber slides and fixed and parenthesis indicate percentage of cells positive for stained at 24 h postinfection, except baby hamster capsid antigen as determined by the indirect immukidney and rat embryo fibroblasts, which were fixed at nofluorescence test. Each point represents the average 48 h postinfection. of five determinations. Symbols: (A) SE3049; (U) b+, Fluorescence; -, fluorescence negative. ips.
Rat
120
J. VIROL.
TACHOVSKY AND HARE
greater amount of antigenic material in 3049infected cells. Radioimmunoassay of capsid proteins in subcellular fractions. The demonstration of more antibody-binding material in 3049infected cells supported the hypothesis that more capsid polypeptide was synthesized. An attempt was made to demonstrate that more polypeptide was present in the cytoplasmic fraction of infected cells. Table 4 shows the results obtained by indirect radioimmunoassay using the nuclear and cytoplasmic fractions of virus-infected cells. In each case 3049-infected cell fractions, both nuclear and cytoplasmic, bound more '25I-antiglobulin than the lpS fractions. Significantly more antigen was detectable in the cytoplasmic fraction of the 3049-infected cells by the anti-disaggregated virus antisera than by the anti-whole virus antiserum. The nuclear fractions of both infected cell populations contained detectable viral antigen, the reacting species present in a form recognizable by both the anti-whole virus antiserum and the anti-disaggregated virus antisera. Inhibition of capsid protein synthesis by metabolic inhibitors. The effect of several metabolic inhibitors on the synthesis of capsid proteins as measured by the indirect radioimmunoassay in 3049 virus-infected cells was then studied. As shown in Fig. 3, fluorodeoxyuridine, actinomycin D, a-amanitin and cycloheximide all strongly inhibited synthesis of the antibodybinding protein when added 1 h after infection, followed by a progressive reduction in the degree of inhibition during the course of infection. Several interesting points should be noted. The synthesis of capsid antigen was as dependent on continuing DNA synthesis as it was on RNA synthesis. The effect of ca-amanitin, an inhibitor
*FdU
800
ACYCLOHEXIMIDE
~605040-
05 3020-
O-'4,.......
100
5
10
15
20
25
30
TIME OF ADDITION OF INHIBITOR (Hrs post-infection) FIG. 3. The effect of various metabolic inhibitors on the synthesis of capsid proteins in 3049-infected mouse embryo cells as measured by binding of 125J_ antiglobulin to cells treated with anti-3049 pool antiserum. The inhibitors were added at different times after infection and all cover slips were harvested at 30 h postinfection. Results are expressed as a percentage of binding to control, untreated cells at 30 h and are charted at the time when the inhibitor was added. Actinomycin D, 0.05 ,ug/ml; a-amanitin, 1 Asg/ml; fluorodeoxyuridine (FdU), 15 A.g/ml; cycloheximide, 1 lAg/ ml. Five duplicate cover slip samples were assayed for each determination.
of nucleoplasmic RNA polymerase II (18), was lost more rapidly than that of actinomycin D, in spite of the fact that it was fully as active as actinomycin D when added early in infection. Finally, as would be expected, cycloheximide inhibited synthesis of the protein for a longer time than any of the other compounds.
DISCUSSION
The 3049 strain of Py virus displays a uniquely different phenotype from other reported strains in that capsid polypeptide is TABLE 4. Subcellular distribution of capsid detectable in the cytoplasm as well as the polypeptide in IpS- and 3049-infected mouse embryo nucleus of the infected mouse cell. Several radioimmunoassay cells as determined by indirect possible mechanisms for this phenotype were considered, namely, altered diffusion of capsid Counts/min per welIb protein from cytoplasm to nucleus, or enhanced Anti-3049 Anti-3049D Anti l SD synthesis of capsid protein (6). This study was Antigena carried out to measure the relative amounts of capsid polypeptide in the whole infected cell or 210 + 9 210 + 8 SE 3049/cytoplasm 60 ± 2 0 0 40 ± 2 fractions thereof, following infection with the lpS/cytoplasm SE 3049/nucleus 550 ± 15 730 ± 16 400 + 6 3049 virus and a control, wild-type virus (1pS) 280 ± 11 320 ± 9 170 4 3 lpS/nucleus using more quantitative, radioimmunoassay techniques. aCells were cultured in 100-mm petri dishes inAntisera to intact virions as well as the fected with virus at a multiplicity of 40 PFU/cell, and dissociated, S-carboxymethylated polypeptides harvested 24 h later. bMean ±e 1 standard deviation for three determina- derived from purified virions were prepared and characterized by several different criteria. Antitions.
VOL. 16, 1975
serum to intact 3049 virions detected a major common antigenic determinant on the 3049 and lpS particle but did not react with the dissociated polypeptides. Antisera to the disaggregated, S-carboxymethylated polypeptides of either 3049 or lpS virions detected a major common antigenic determinant on the dissociated polypeptides of both virus strains. These sera did not react with the intact virion of either strain as measured by neutralization of infectivity, inhibition of hemagglutination, or formation of precipitin bands. These data indicated that the capsid polypeptides of the two virus strains were immunochemically indistinguishable and therefore the use of a radioimmunoassay to measure the relative quantities of capsid proteins in the cells infected with the two virus strains was valid. Furthermore, these reagents could be utilized to identify precursor polypeptides as well as the protein configuration characteristic of intact virions. With these reagents it was then shown that the cytoplasm as well as the nucleus of the 3049-infected cell contained virus-specific polypeptides that reacted with antisera to both intact virions and the dissociated polypeptides. The lpS-infected cell, however, showed evidence for the presence of polypeptides carrying the two antigenic determinants only in the nucleus. These findings support the concept that the capsid polypeptide is synthesized in the cytoplasm and is rapidly translocated to the nucleus in the wild-type infection where particle assembly takes place. The precursor molecule, however, never attains a level sufficient to be observed in the cytoplasm with the indirect fluorescent antibody technique. In the case of the 3049 strain, infection is associated with the production of the polypeptide resulting in a concentration in the cytoplasm sufficient to react positively with the fluorescent antibody technique. Furthermore, the demonstration that the antigenic determinants characteristic of both intact virions and precursor polypeptides are found in the cytoplasm as well as the nucleus suggests that some structural maturation may also take place in the cytoplasm. The next step was to measure the relative quantity of virus-specfic antigenic material in cells infected with 3049 and lpS viruses using an indirect radioimmunoassay as described by Hayashi et al. (16). By using carefully controlled infectious doses of each virus strain to yield equal numbers of cells producing capsid protein detected by immunofluorescence, it was possible to demonstrate that 3049-infected cells bind several-fold more virus-specific antiserum than lpS-infected cells. This was detected by
POLYOMA CAPSID PROTEIN SYNTHESIS
121
the specific binding of 125I-labeled IgG fraction of goat anti-rabbit gamma globulin. Furthermore, the cytoplasmic fraction of the 3049infected cell was shown to contain antibodybinding antigen under conditions in which the cytoplasm of lpS-infected cells failed to bind antibody. These findings provide direct evidence that the synthesis of capsid protein is significantly higher in 3049- than in lpS-infected cells and that there is a significant accumulation of capsid polypeptide precursor molecules in the cytoplasm of the 3049-infected cell. Recently it has been demonstrated that there is also a twofold greater quantity of virus-specific polyadenylated RNA in both the nuclear RNA and polyribosomal fractions of 3049-infected cells as compared to those infected with lpS virus (22). On this basis, then, it can be proposed that the phenotype of the 3049 virus, namely, the presence of capsid polypeptide molecules in the cytoplasm, is the direct result of the increased numbers of mRNA templates specifying at least the major structural protein, VP2 (21), rather than some virus controlled enhancement of the translation rate from the mRNA. Direct -assessment of the rate of translation or the quantity of translatable mRNA has not been carried out, however. Two other findings bear on the question as to which level of genetic control is altered in the 3049 virus replication process. The first is the demonstration that the expression of the phenotype is closely correlated with the presence of certain mouse chromosomes such that in the absence of mouse genetic information, i.e., in hamster cells, the phenotype is suppressed. On the other hand, in mouse-hamster hybrids with sufficient mouse DNA represented, the virus replicates and the phenotype is expressed. The rat embryo cell is of interest in that a significant number of cells are permissive for the expression of "late" functions and the 3049 phenotype is expressed. These findings suggest that some host cell function is intimately associated with the expression of the phenotype. A second finding of interest is the prolonged sensitivity of the synthesis of capsid protein to inhibition of DNA synthesis by FdU, with a pattern quite similar to that produced by actinomycin D (Fig. 3). This is reminiscent of the demonstration by Glover (10) that inhibition of Py DNA synthesis restricts the quantity and quality of the viral RNA transcribed "late" in infection to yield an RNA population that resembles "early" transcripts. Taken together, these findings suggest that the rate of synthesis of this late virus gene
122
TACHOVSKY AND HARE
product, the major capsid protein, is regulated by the quantity of mRNA available for translation. Furthermore, the quantity of mRNA may in turn be controlled by some host cell function which determines the rate of transcription of the viral genome following the onset of viral DNA synthesis. This function could be either a host DNA or RNA polymerase the function of which is modified by some factor associated with the synthesis of late viral functions. The reverse transcription co-factor postulated by Amati (1) would also fit with this postulate. The elucidation of the mechanism of this unique phenotype should provide a significant increase in the understanding of genetic regulation in eukaryotic host cell-virus systems. LITERATURE CITED 1. Amati, P. 1974. A working model for oncogenic DNA virus replication. J. Theor. Biol. 46:221-227. 2. Bale, W. F., R. W. Helmkamp, T. P. Davis, M. J. Izzo, R. L. Goodland, M. A. Contreras, and I. L. Spar. 1966. High specific activity labeling of protein with 131I by the iodine monochloride method. Proc. Soc. Exp. Biol. Med. 122:407-414. 3. Basilico, C., Y. Matsuya, and H. Green. 1970. The interaction of polyoma virus with mouse-hamster hybrid cells. Virology 41:295-305. 4. Benjamin, T. L. 1970. Host range mutants of polyoma virus. Proc. Natl. Acad. Sci. U.S.A. 67:390-399. 5. Bereczky, E., R. Hughes, J. M. Bowen, W. Munyon, and L. Dmochowski. 1965. Study of DNA synthesis and antigen formation in polyoma virus-infected mouse embryo cells by autoradiography and immunofluorescence. Texas Rep. Biol. Med. 23:3-15. 6. Betts, R. F., T. G. Tachovsky, and J. D. Hare. 1972. Studies on the mechanism of cytoplasmic antigen accumulation following infection with a new variant of polyoma virus. J. Gen. Virol. 16:29-38. 7. Crawford, L. V. 1962. The adsorption of polyoma virus. Virology 18:177-181. 8. Diamond, L., and L. V. Crawford. 1964. Some characteristics of large-plaque and small-plaque lines of polyoma virus. Virology 22:235-244. 9. Eckhart, W. 1969. Complementation and transformation by temperature-sensitive mutants of polyoma virus. Virology 38:120-125. 10. Glover, D. M. 1974. Coupling of polyoma DNA and RNA synthesis. Biochem. Biophys. Res. Commun. 57:1137-1143. 11. Hare, J. D., P. Balduzzi, and H. R. Morgan. 1963.
J. VIROL.
12. 13.
14.
15. 16. 17.
18.
19.
20.
21. 22.
23. 24.
25. 26.
Polyoma virus and L cell relationship. I. Some characteristics of a cell line persistently infected with polyoma virus. J. Natl. Cancer Inst. 30:45-56. Hare, J. D., and J. Chan. 1966. Antigenic variation among polyoma viruses from different sources demonstrated by plaque neutralization. Virology 30:62-73. Hare, J. D. 1967. Localization and characterization of the phenylalanine transport mechanism in normal and polyoma-transformed hamster cells. Cancer Res. 27:2357-2363. Hare, J. D. 1967. Transplant immunity to polyomainduced tumor cells. IV. A polyoma strain defective in transplant antigen induction. Virology 31:625-632. Hare, J. D. 1970. A new type of variation among polyoma virus characterized by cytoplasmic accumulation of capsid antigen. Virology 40:978-988. Hayashi, K., J. Rosenthal. and A. L. Notkins. 1972. Iodine-125 labeled antibody to viral antigens binding to the surface of virus-infected cells. Science 176:516-518. Henle, G., F. Deinhardt, and J. Rodrigues. 1959. The development of polyoma virus in mouse embryo cells as revealed by fluorescent antibody staining. Virology 8:388-391. Lindell, T. J., F. Weinberg, P. W. Morris, R. G. Roeder, and W. J. Rutter. 1970. Specific inhibition of nuclear RNA polymerase II by a-amanitin. Science 170:447-449. Malmgren, R. A., R. Rabatti, and A. S. Rabson. 1960. Intracellular localization of polyoma virus antigen demonstrated with fluorescein labelled antisera. J. Natl. Cancer Inst. 24:581-588. McCulloch, E. A., A. F. Howatson, L. Siminovitch, A. A. Axelrad, and A. W. Ham. 1959. Tumor induction in Swiss mice by virus obtained originally from a mammary tumor in C3H/Jax mouse and then cultivated in vitro. Proc. Am. Assoc. Cancer Res. 3:41. Roblin, R., E. Harle, and R. Dulbecco. 1971. Polyoma virus proteins. I. Multiple virion components. Virology 45:555-566. Rutherford, R. B., and J. D. Hare. 1974. Evidence for a regulatory function related to the expression of the polyoma genome following the onset of virus DNA replication. Biochem. Biophys. Res. Commun 58:839-846. Sachs, L., and M. Fogel. 1960. Polyoma virus synthesis in tumor cells as measured by the fluorescent antibody technique. Virology 11:722-736. Williams, M. G., and R. Sheinin. 1961. Cytological studies of mouse embryo cells infected with polyoma virus using acridine orange and fluorescent antibody. Virology 13:368-369. Vestergard-Jorgensen, P. E. 1972. Freund's adjuvants: their influence on the specificity of viral antisera. Acta Pathol. Microbiol. Scand. Sect. B 80:931-933. Winocour, E. 1963. Purification of polyoma virus. Virology 19:158-168.
