JOURNAL
OF
VIROLOGY, Nov. 1979, p. 640-647
Vol. 32, No. 2
0022-538X/79/11-0640/08$02.00/0
In Vitro Reassembly of Infectious Polyoma Virionst J. N. BRADY,: J. D. KENDALL, AND R. A. CONSIGLI* Section of Virology and Oncology, Division of Biology, Kansas State University, Manhattan, Kansas 66506
Received for publication 30 April 1979
Initial experiments in our laboratory have successfully reassembled infectious polyoma virions from dissociated virion products. Virions treated with ethyleneglycol-bis-N,N'-tetraacetic acid and the reducing agent ,B-mercaptoethanol at pH 7.5 were dissociated to a 48S DNA-protein complex and capsomere subunits. The virion dissociation products were not infectious by plaque assay and lacked hemagglutination activity. These virion dissociation products were reassembled to intact virions by overnight dialysis against a reassembly buffer containing CaCl2, dimethyl sulfoxide, and Triton X-100 in phosphate-buffered saline at pH 7.4. The biophysical characteristics of the reassembled virions were identical to those of untreated virions in that the reassembled virions had a sedimentation value of 240S in sucrose gradients and a buoyant density of 1.315 g/cm: in CsCl isopycnic gradients. The reassembled virions were intact as determined by electron microscopy and were found to be 60% resistant to DNase I treatment. Biologically, the reassembled purified virions were found to partially regain both hemagglutinating activity and plaque-forming ability.
The successful in vitro reassembly of various RNA phages (24, 27, 32), plant viruses (1, 13-15, 21, 22, 25, 34), and poliovirus (10) suggest that assembly of these virions involves, at least in part, self-assembly mechanisms. In vitro reassembly of tobacco mosaic virus has increased the knowledge of the nucleic acid-protein interactions of the virions and tobacco mosaic virus viral morphogenesis tremendously (14). No reassembly system for obtaining infectious virus has been reported for any mammalian DNA virus. However, the simplicity of polyoma virions would appear to make this virus ideally suited for such in vitro reassembly studies. The structure of polyoma virus was described in detail in a recent review by Finch and Crawford (12). The virus contains a single supercoiled circular DNA of approximately 3.4 x 106 daltons encapsidated in an icosahedral protein shell. Three proteins, VPI, VP2 and VP:3, which are coded for by the viral DNA, and four cellular histones, VP4, VP5, VP6, and VP7, are found in the mature virion (3, 8, 9, 11, 16, 18, 20, 30, 31, 33). Friedmann has attempted the in vitro reassembly of alkaline carbonate-disrupted polyoma virions (17). Similar studies have been reported by Christensen and Rachmeler with simian virus 40 (5). Recently, Aposhian's laboratory has ret Contribution no. 79-346-j from the Kansas Agricultural Experiment Station, Kansas State University. t Present address: Laboratory of Biology and Viruses, National Institute of Allergy and Infectious Diseases, Bethesda,
MD 20205.
640
ported a cell-free system which assembles 106 daltons of DNA into polyoma capsids (K. Keck, S. M. Barr, and H. V. Aposhian, Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, S74, p. 225; personal communication). In all cases, the reassembled particles either were biophysically dissimilar from complete virions or were not infectious. The data presented in this paper demonstrate that complete polyoma virions, which are biophysically and biologically identical to mature virions, may be reassembled after dissociation of polyoma virions to a DNA-protein complex and capsomere subunits by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid (EGTA) and 83-mercaptoethanol (ME) at pH 7.5. MATERIALS AND METHODS Cell and virus propagation. The preparation of primary cultures of mouse embryo and mouse kidney cells has been described (7, 35). For roller-bottle cell cultures, each bottle was seeded with 100 ml of a cellmedia mixture containing 2 x 10" cells per ml, prepared as described above. Wild-type polyoma virus was used to infect cells at a multiplicity of infection of 10. Infected cultures were maintained in serum-free Dulbecco-modified Eagle medium (29). Virus purification. Virus was purified from the infected cell lysate either as described previously for small volumes (29) or for large volumes as described by Friedmann and Haas (19) by polyethylene glycol precipitation. The CsCl gradients used to purify the virus were prepared as described by Brunck and Leick (4) and described in greater detail previously (2, 3). Preparation of radioactivity labeled polyoma
VOL. 32, 1979
REASSEMBLY OF INFECTIOUS POLYOMA VIRIONS
virions. The preparation of [3H]thymidine-labeled polyoma virions has been previously described (3, 29). Dissociation of polyoma virions. Stock solutions of each component of the dissociation buffer (Tris, EGTA, NaCI) were prepared and stored at 4°C, with the exception of ME, which was prepared fresh for each assay. Optimum virion dissociation was obtained when ME, NaCl, and EGTA were added in this order. Specific experimental conditions for polyoma dissociation are described in each experiment. All dissociation reaction mixtures were incubated at room temperature.
Quantitative assay. Cesium chloride densities were determined from the refractive index using a Bausch and Lomb refractometer and calculated using the equation of Vinograd and Hearst (36). Radioactivity was quantitated in a toluene-Triton (3:1) scintillation fluid using a Beckman LS-233 liquid scintillation counter. Velocity sedimentation. Polyoma virions, reassembled virions, or products derived from dissociated virions were layered onto 10 to 30% (wt/vol) sucrose gradients containing 0.15 M NaCl-0.01% Triton X-100 (Rutger Chemical Co.) in Tris-hydrochloride buffer (0.01 M, pH 7.4). Centrifugation was in an SW50.1 rotor at 40,000 rpm (4°C) for 40 min. Sedimentation coefficients were determined by comparison with 240S polyoma virions. Buoyant density determinations of polyoma virions and reassembled polyoma virions. Both polyoma virions and reassembled virions were isolated from sucrose gradients as described above. Preformed CsCl gradients containing 1.0 ml of 1.35-g/cm3, 2.0 ml of 1.32-g/cm3, and 1.5 ml of 1.29-g/cm3 CsCl concentrations were loaded with 0.5 ml of the respective virus sample. The samples were then centrifuged to equilibrium in an SW50.1 rotor at 35,000 rpm for 20 h at 10°C. Fractions were collected and assayed for radioactivity and density determinations. Electron microscopy. Samples were mounted on carbon-coated grids and stained with freshly prepared 1% uranyl acetate. Specimens were examined with a Philips 201 electron microscope operated at 60 kV. Reassembly of polyoma virions. After EGTAME dissociation of polyoma virions to a DNA-protein complex and capsomere subunits, Triton X-100 was added to a final concentration of 0.01%. CaCl2 was then added to a final concentratiion of 5 x 10-4 to 5 X 10-7 M (determined for each virion preparation), and samples were mixed and allowed to incubate at room temperature for 10 min. Dimethyl sulfoxide (DMSO) was added gradually to a final concentration of 20%, and samples were mixed and allowed to incubate for 30 min at room temperature. At the end of the incubation period, the samples were dialyzed against a reassembly buffer containing appropriate CaCl2 (determined for each virion preparation), 10% DMSO, and 0.01% Triton X-100 in phosphate-buffered saline (pH 7.4) ovemight at 4°C. After dialysis, the reassembled virion samples were passed gently three times through a needle and syringe to break any viral aggregates and layered onto 10 to 30% sucrose gradients for analysis. HA, fluorescent antibody, and plaque assays. The fluorescent antibody, hemagglutination (HA), and
641
plaque assays for polyoma virus have been described previously (6, 7, 31).
RESULTS Before virion reassembly studies were initiated, biophysical and biological criteria were established for both EGTA-ME-dissociated virions and reassembled virions. After dissociation of radioactively labeled virions by treatment with EGTA-ME, the samples were analyzed by velocity sedimentation in sucrose gradients, and the 240S region (sedimentation value of intact polyoma virions) was analyzed to determine that no radioactivity, HA activity, or plaque-forming ability remained. In addition, a portion of the dissociated virion preparation was treated with pancreatic DNase I to insure the complete susceptibility of the DNA-protein complex to DNase. The dissociated virion preparation was also examined by electron microscopy to insure that only capsomeres and DNA-protein complex were present. Before dissociated virion preparations were subjected to reassembly conditions, each of the above criteria was examined critically. Any dissociated virion preparation that did not meet all the above criteria indicated that inadequate dissociation of the virions had occurred, and the sample was discarded. We feel that by meeting these standard dissociation requirements, the possibility of partially dissociated virions "snapping back" would be minimized. After virion reassembly, the above criteria were again analyzed to determine whether the reassembly of polyoma virions had occurred. Optimization of reassembly buffer. A preparation of [3H]thymidine-labeled virions was dissociated by treatment with 0.1 M ME0.15 M NaCl-I mM EGTA in Tris-hydrochloride buffer at pH 7.4. After dissociation, optimum concentrations of ingredients found in the reassembly buffer were determined. The optimum NaCl concentration for the reassembly buffer was 0.15 M as determined over a range of 0.075 M to 0.17 M NaCl. The optimum pH for polyoma virion reassembly was 7.4 as determined over a range of 6.5 to 8.0. The percentage of reassembled virions decreased significantly at pH values above 7.4. The addition of DMSO to 20% after dissociation and dialysis against reassembly buffer with 10% DMSO was found to give the most reproducible reassembly results (data not shown). The proper Ca2+ concentration was extremely critical for virion reassembly, and we found that the Ca2+ concentration to be used was variable depending upon the amount of Ca2" originally associated with the virus preparation prior to dissociation. Thus, each polyoma preparation that was dissociated underwent a Ca2" titration
642
BRADY, KENDALL, AND CONSIGLI
J. VIROL.
to determine the concentration of Ca2" to be used in the reassembly buffer. Figure 1 illustrates such an experiment, where dissociated virions were dialyzed overnight against reassembly buffer containing various concentrations of Ca2". Polyoma virus reassembly was analyzed by velocity sedimentation in sucrose gradients. Dissociated virions, which were not dialyzed against reassembly buffer, demonstrated that all the virions were dissociated since all of the
[3H]thymidine-labeled polyoma DNA-protein complex sedimnented at 48S with no labeled virions remaining in the 240S region (arrow) of the gradient (Fig. 1A). No HA or plaque-forming activity could be detected in the 240S region of 80
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20
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20
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FRACTION NUMBERS
FIG. 1. Velocity sedimentation of EGTA-ME-dissociated and reassembled polyoma virions. ['HAthymidine-labeled virions were exposed to I mM EGTA0.1I M ME-0. 15 M NaCl in 0.05 M Tris buffer (pH 7.4) for 30 min at room temperature. After dissociation, the sample was divided into four equal portions and treated as follows: (A) dissociated virion control; the remaining dissociated virion samples were dialyzed overnight against 10%/ DMSO-0.01% Triton X-100 in phosphate-buffered saline (pH 7.4) with (B) 5 x i0-' M CaCl2, (C) 5 X 10-i; M CaCl2, and (D) 5 x 10-7 M CaCI2. Samples were then layered onto a 10 to 30%,o sucrose gradient and centrifuged in an SW50.1 rotor at 40,000 rpm for 40 min at 40C. Radioactivity determinations were made on the fractions, and the remainder was stored at 40C for further analysis. Arrow indicates sedimentation position of 240S polyoma virions centrifuged in a parallel tube. All samples were labeled with [3H]thymidine.
the gradient. However, when the dissociated virions were dialyzed against reassembly buffer containing various concentrations of Ca2", it was found that dissociated virions were reassembled to varying degrees and sedimented in the 240S region of the gradient. When dissociated polyoma virions were dialyzed against reassembly buffer containing 5 x 10-5 M Ca2", 17% of the radioactive counts in the gradient were found in the 240S region (Fig. 1B), whereas 5 x 10-6 M Ca2" allowed 22% (Fig. 1C) and 5 x 10-7 M Ca2" allowed 33% reassembly (Fig. 1D). Apparently, higher concentrations of Ca2" are inhibitory for polyoma reassembly. Complete removal of Ca2" from the reassembly buffer was also found to decrease reassembly of polyoma virions. Infectivity of the respective reassembled virion preparations will be described subsequently. Susceptibility of reassembled virions to DNase. After dissociation, a sample of the dissociated virion preparation was analyzed by velocity sedimentation in sucrose gradients before and after treatment with pancreatic DNase 1 (Fig. 2). Prior to DNase treatment, only one peak of radioactivity was found, corresponding to the 48S DNA-protein complex, in the dissociated virion preparation (Fig. 2A). When the dissociated virion preparation was subjected to DNase treatment before centrifugation, only soluble DNA fragments could be seen at the top of the gradient (Fig. 2B). After dialysis of the dissociated virion sample against the reassembly buffer, the samples were again analyzed by sedimentation velocity sucrose gradients before and after treatment with pancreatic DNase. Prior to DNase treatment, the reassembled virion sample was found to contain not only a large peak of radioactivity in the region of polyoma complete virions, but also, apparently, many species of intermediate particles which had not reached completion of reassembly (Fig. 2A). Particles with a sedimentation value of 48S (DNA-protein complex) to 240S (polyoma completes) were evident in the gradient profile. After DNase treatment of the reassembled virion preparation, only one peak of radioactivity, which sedimented at 240S, was detected in the gradient (Fig. 2B). From the radioactivity present in the region of 240S in Fig. 2A and B, it was estimated that 60% of the reassembled 240S species was resistant to DNase treatment. Conversely, virtually all of the intermediate reassembled products (fractions 15 to 30) were susceptible to DNase treatment. Buoyant density analysis of reassembled polyoma virions. To further examine the biophysical properties of the reassembled polyoma virions, fractions 11 to 15 (Fig. 2) from the sucrose gradients (240S region) were pooled and
643
REASSEMBLY OF INFECTIOUS POLYOMA VIRIONS
VOL. 32, 1979
region of reassembled polyoma virions (data not shown). The results of these experiments indicate that the particles sedimenting at 240S in the sucrose gradients were likely virions and not aggregates of DNA-protein complex and capsomere subunits which just fortuitously sedimented at 240S. In addition, the reassembled virions (with or without DNase treatment) would appear to be stable to CsCl. Little or no radioactivity was found in the pellet of the CsCl gradient, where free DNA or DNA-protein complex would be found. Electron microscopy of intact, dissociated, and reassembled polyoma virions. The reassembly of polyoma virions was also examined by electron microscopy. Polyoma virions before dissociation can be seen in Fig. 4A. After dissociation of the virions with EGTAME, the virions were reduced to free DNA-protein complexes and capsomere subunits (Fig. 4B). No intact virions or partially dissociated virions could be detected in the dissociated vir-
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FIG. 2. Velocity sedimentation of EGTA-ME-dissociated and reassembled polyoma virions after treatment with DNase 1. [3H]thymidine-labeledpolyoma virions were exposed to 1 mM EGTA-0.1 M ME-0.15 M NaCl in 0.05 M Tris buffer (pH 7.4) for 30 min at room temperature. The dissociated virion sample was then divided into two equal portions. The dissociated virion control sample was stored at 4°C. The remaining sample was dialyzed against 5 x 10-' M CaCl2-10% DMSO-0.01% Triton X-100 in phosphate-buffered saline (pH 7.4). A portion of the dissociated virion and reassembled virion preparations was treated with 50 yg ofpancreatic DNase 1 per ml 30 min at 37°C. Samples were layered onto 10 to 30%o sucrose gradients and centrifuged in an SW50.1 rotor at 40,000 rpm for 40 min at 4'C. Radioactivity determinations were made on the fractions, and the remainder was stored at 4°C for further analysis. (A) No DNase treatment; (B) DNase treated. (O EGTAME-dissociated polyoma virions; (0) reassembled polyoma virions.
analyzed by equilibrium CsCl centrifugation (Fig. 3). Control polyoma virions banded at a density of 1.315 g/cm3. The reassembled virions, with or without DNase treatment, were also found to have a density of 1.315 g/cm3. In similar experiments, in which fractions 11 to 15 were pooled from dissociated virion sucrose gradients, no radioactivity could be detected in the density
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40 30 20 FRACTION NUMBER FIG. 3. Buoyant density determination ofpolyoma virions and reassembled polyoma virions in CsCl gradients. Polyoma virions and reassembled virions (with and without DNase I treatment) were isolated from sucrose gradients as shown in Fig. 2. Samples were layered onto preformed CsCl gradients and centrifuged in an SW50.1 rotor at 35,000 rpm for 20 h at 10°C. Fractions were collected and assayed for radioactivity and density determinations. Arrow indicates density of control polyoma virions. Symbols: (0) reassembled polyoma virions, no DNase treatment; (0) reassembled polyoma virions, DNase treated.
644
BRADY, KENDALL, AND CONSIGLI
J. VIROL.
IIP A
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FIG. 4. Electron microscopy of intact, EGTA-ME-dissociated, and reassembled polyoma virions. (A) Control; intact polyoma virions; (B) EGTA-ME-dissociated polyoma virions; (C) reassembled polyoma virions; (D) reassembled polyoma virions isolated from 240S region of sucrose gradient as shown in Fig. 2. Samples were mounted on specimen grids, stained with uranyl acetate, and examined. x180,000.
ion preparations. Following dialysis of the dissociated virion preparation against the reassembly buffer, however, a mixture of virions, DNAprotein complexes, and capsomere subunits could be visualized in the electron microscope (Fig. 4C). As an additional control to insure that the particles shown in Fig. 4C were indeed intact virions, the sample was centrifuged on velocity sedimentation sucrose gradients as shown in Fig. 1, and the 240S region was isolated. Samples of the reassembled 240S particles were then examined by electron microscopy. Polyoma virions identical to those before dissociation were found in the isolated 240S fraction (Fig. 4D). Thus, if one compares the biophysical properties of intact, dissociated, and reassembled polyoma virions, the intact virions and reassembled virions are virtually identical, whereas the dissociated virions are markedly different (Table 1). The only significant difference between the intact and reassembled virions was the partial sensitivity of the reassembled virions to DNase treatment. Approximately 40% of the reassembled 240S virions were sensitive to DNase treat-
ment. However, the reassembled virions resistant to DNase treatment would appear to be quite "tight," since 93% of the DNA found inside these virions was found to be component I polyoma DNA (data not shown).
Analysis of hemagglutinating activity and plaque-forming ability of reassembled polyoma virions. Since the reassembled viri.ons appeared similar to intact virions by biophysical examination, the next series of experiments were performed to determine the biological properties of the reassembled virions. After the isolation of the 240S region from sucrose gradients containing intact virions, dissociated virions, and reassembled virions, the three samples were assayed for HA activity, infectivity by the immunofluorescent antibody assay, and plaque-forming ability (Table 2). When the three preparations were assayed for HA activity, the control untreated virions (amount of virus applied to gradient was equal to that added to dissociation mix) had an HA titer of 5,120 HA units per ml, whereas the reassembled virions had a titer of 640 HA units per ml. The dissoci-
REASSEMBLY OF INFECTIOUS POLYOMA VIRIONS
VOL. 32, 1979
645
TABLE 1. Comparison of biophysical properties of intact, dissociated, and reassembled polyoma virions % Compo% DNase Density Virion sample
Intact Dissociated
Reassembled
S2.
(sucrose)
240 48S complex; 18, 12, 5S capsomeres 240
TABLE 2. Comparison of biological properties of intact, dissociated, and reassembled polyoma virions PFU/ml HA titer FAM Virion sample (+) 3 x 108 5,120 Intact 0 (-)
OF
VIROLOGY, Nov. 1979, p. 640-647
Vol. 32, No. 2
0022-538X/79/11-0640/08$02.00/0
In Vitro Reassembly of Infectious Polyoma Virionst J. N. BRADY,: J. D. KENDALL, AND R. A. CONSIGLI* Section of Virology and Oncology, Division of Biology, Kansas State University, Manhattan, Kansas 66506
Received for publication 30 April 1979
Initial experiments in our laboratory have successfully reassembled infectious polyoma virions from dissociated virion products. Virions treated with ethyleneglycol-bis-N,N'-tetraacetic acid and the reducing agent ,B-mercaptoethanol at pH 7.5 were dissociated to a 48S DNA-protein complex and capsomere subunits. The virion dissociation products were not infectious by plaque assay and lacked hemagglutination activity. These virion dissociation products were reassembled to intact virions by overnight dialysis against a reassembly buffer containing CaCl2, dimethyl sulfoxide, and Triton X-100 in phosphate-buffered saline at pH 7.4. The biophysical characteristics of the reassembled virions were identical to those of untreated virions in that the reassembled virions had a sedimentation value of 240S in sucrose gradients and a buoyant density of 1.315 g/cm: in CsCl isopycnic gradients. The reassembled virions were intact as determined by electron microscopy and were found to be 60% resistant to DNase I treatment. Biologically, the reassembled purified virions were found to partially regain both hemagglutinating activity and plaque-forming ability.
The successful in vitro reassembly of various RNA phages (24, 27, 32), plant viruses (1, 13-15, 21, 22, 25, 34), and poliovirus (10) suggest that assembly of these virions involves, at least in part, self-assembly mechanisms. In vitro reassembly of tobacco mosaic virus has increased the knowledge of the nucleic acid-protein interactions of the virions and tobacco mosaic virus viral morphogenesis tremendously (14). No reassembly system for obtaining infectious virus has been reported for any mammalian DNA virus. However, the simplicity of polyoma virions would appear to make this virus ideally suited for such in vitro reassembly studies. The structure of polyoma virus was described in detail in a recent review by Finch and Crawford (12). The virus contains a single supercoiled circular DNA of approximately 3.4 x 106 daltons encapsidated in an icosahedral protein shell. Three proteins, VPI, VP2 and VP:3, which are coded for by the viral DNA, and four cellular histones, VP4, VP5, VP6, and VP7, are found in the mature virion (3, 8, 9, 11, 16, 18, 20, 30, 31, 33). Friedmann has attempted the in vitro reassembly of alkaline carbonate-disrupted polyoma virions (17). Similar studies have been reported by Christensen and Rachmeler with simian virus 40 (5). Recently, Aposhian's laboratory has ret Contribution no. 79-346-j from the Kansas Agricultural Experiment Station, Kansas State University. t Present address: Laboratory of Biology and Viruses, National Institute of Allergy and Infectious Diseases, Bethesda,
MD 20205.
640
ported a cell-free system which assembles 106 daltons of DNA into polyoma capsids (K. Keck, S. M. Barr, and H. V. Aposhian, Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, S74, p. 225; personal communication). In all cases, the reassembled particles either were biophysically dissimilar from complete virions or were not infectious. The data presented in this paper demonstrate that complete polyoma virions, which are biophysically and biologically identical to mature virions, may be reassembled after dissociation of polyoma virions to a DNA-protein complex and capsomere subunits by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid (EGTA) and 83-mercaptoethanol (ME) at pH 7.5. MATERIALS AND METHODS Cell and virus propagation. The preparation of primary cultures of mouse embryo and mouse kidney cells has been described (7, 35). For roller-bottle cell cultures, each bottle was seeded with 100 ml of a cellmedia mixture containing 2 x 10" cells per ml, prepared as described above. Wild-type polyoma virus was used to infect cells at a multiplicity of infection of 10. Infected cultures were maintained in serum-free Dulbecco-modified Eagle medium (29). Virus purification. Virus was purified from the infected cell lysate either as described previously for small volumes (29) or for large volumes as described by Friedmann and Haas (19) by polyethylene glycol precipitation. The CsCl gradients used to purify the virus were prepared as described by Brunck and Leick (4) and described in greater detail previously (2, 3). Preparation of radioactivity labeled polyoma
VOL. 32, 1979
REASSEMBLY OF INFECTIOUS POLYOMA VIRIONS
virions. The preparation of [3H]thymidine-labeled polyoma virions has been previously described (3, 29). Dissociation of polyoma virions. Stock solutions of each component of the dissociation buffer (Tris, EGTA, NaCI) were prepared and stored at 4°C, with the exception of ME, which was prepared fresh for each assay. Optimum virion dissociation was obtained when ME, NaCl, and EGTA were added in this order. Specific experimental conditions for polyoma dissociation are described in each experiment. All dissociation reaction mixtures were incubated at room temperature.
Quantitative assay. Cesium chloride densities were determined from the refractive index using a Bausch and Lomb refractometer and calculated using the equation of Vinograd and Hearst (36). Radioactivity was quantitated in a toluene-Triton (3:1) scintillation fluid using a Beckman LS-233 liquid scintillation counter. Velocity sedimentation. Polyoma virions, reassembled virions, or products derived from dissociated virions were layered onto 10 to 30% (wt/vol) sucrose gradients containing 0.15 M NaCl-0.01% Triton X-100 (Rutger Chemical Co.) in Tris-hydrochloride buffer (0.01 M, pH 7.4). Centrifugation was in an SW50.1 rotor at 40,000 rpm (4°C) for 40 min. Sedimentation coefficients were determined by comparison with 240S polyoma virions. Buoyant density determinations of polyoma virions and reassembled polyoma virions. Both polyoma virions and reassembled virions were isolated from sucrose gradients as described above. Preformed CsCl gradients containing 1.0 ml of 1.35-g/cm3, 2.0 ml of 1.32-g/cm3, and 1.5 ml of 1.29-g/cm3 CsCl concentrations were loaded with 0.5 ml of the respective virus sample. The samples were then centrifuged to equilibrium in an SW50.1 rotor at 35,000 rpm for 20 h at 10°C. Fractions were collected and assayed for radioactivity and density determinations. Electron microscopy. Samples were mounted on carbon-coated grids and stained with freshly prepared 1% uranyl acetate. Specimens were examined with a Philips 201 electron microscope operated at 60 kV. Reassembly of polyoma virions. After EGTAME dissociation of polyoma virions to a DNA-protein complex and capsomere subunits, Triton X-100 was added to a final concentration of 0.01%. CaCl2 was then added to a final concentratiion of 5 x 10-4 to 5 X 10-7 M (determined for each virion preparation), and samples were mixed and allowed to incubate at room temperature for 10 min. Dimethyl sulfoxide (DMSO) was added gradually to a final concentration of 20%, and samples were mixed and allowed to incubate for 30 min at room temperature. At the end of the incubation period, the samples were dialyzed against a reassembly buffer containing appropriate CaCl2 (determined for each virion preparation), 10% DMSO, and 0.01% Triton X-100 in phosphate-buffered saline (pH 7.4) ovemight at 4°C. After dialysis, the reassembled virion samples were passed gently three times through a needle and syringe to break any viral aggregates and layered onto 10 to 30% sucrose gradients for analysis. HA, fluorescent antibody, and plaque assays. The fluorescent antibody, hemagglutination (HA), and
641
plaque assays for polyoma virus have been described previously (6, 7, 31).
RESULTS Before virion reassembly studies were initiated, biophysical and biological criteria were established for both EGTA-ME-dissociated virions and reassembled virions. After dissociation of radioactively labeled virions by treatment with EGTA-ME, the samples were analyzed by velocity sedimentation in sucrose gradients, and the 240S region (sedimentation value of intact polyoma virions) was analyzed to determine that no radioactivity, HA activity, or plaque-forming ability remained. In addition, a portion of the dissociated virion preparation was treated with pancreatic DNase I to insure the complete susceptibility of the DNA-protein complex to DNase. The dissociated virion preparation was also examined by electron microscopy to insure that only capsomeres and DNA-protein complex were present. Before dissociated virion preparations were subjected to reassembly conditions, each of the above criteria was examined critically. Any dissociated virion preparation that did not meet all the above criteria indicated that inadequate dissociation of the virions had occurred, and the sample was discarded. We feel that by meeting these standard dissociation requirements, the possibility of partially dissociated virions "snapping back" would be minimized. After virion reassembly, the above criteria were again analyzed to determine whether the reassembly of polyoma virions had occurred. Optimization of reassembly buffer. A preparation of [3H]thymidine-labeled virions was dissociated by treatment with 0.1 M ME0.15 M NaCl-I mM EGTA in Tris-hydrochloride buffer at pH 7.4. After dissociation, optimum concentrations of ingredients found in the reassembly buffer were determined. The optimum NaCl concentration for the reassembly buffer was 0.15 M as determined over a range of 0.075 M to 0.17 M NaCl. The optimum pH for polyoma virion reassembly was 7.4 as determined over a range of 6.5 to 8.0. The percentage of reassembled virions decreased significantly at pH values above 7.4. The addition of DMSO to 20% after dissociation and dialysis against reassembly buffer with 10% DMSO was found to give the most reproducible reassembly results (data not shown). The proper Ca2+ concentration was extremely critical for virion reassembly, and we found that the Ca2+ concentration to be used was variable depending upon the amount of Ca2" originally associated with the virus preparation prior to dissociation. Thus, each polyoma preparation that was dissociated underwent a Ca2" titration
642
BRADY, KENDALL, AND CONSIGLI
J. VIROL.
to determine the concentration of Ca2" to be used in the reassembly buffer. Figure 1 illustrates such an experiment, where dissociated virions were dialyzed overnight against reassembly buffer containing various concentrations of Ca2". Polyoma virus reassembly was analyzed by velocity sedimentation in sucrose gradients. Dissociated virions, which were not dialyzed against reassembly buffer, demonstrated that all the virions were dissociated since all of the
[3H]thymidine-labeled polyoma DNA-protein complex sedimnented at 48S with no labeled virions remaining in the 240S region (arrow) of the gradient (Fig. 1A). No HA or plaque-forming activity could be detected in the 240S region of 80
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9 98 8 7 76 -6
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20
30
40
10
20
30
40
FRACTION NUMBERS
FIG. 1. Velocity sedimentation of EGTA-ME-dissociated and reassembled polyoma virions. ['HAthymidine-labeled virions were exposed to I mM EGTA0.1I M ME-0. 15 M NaCl in 0.05 M Tris buffer (pH 7.4) for 30 min at room temperature. After dissociation, the sample was divided into four equal portions and treated as follows: (A) dissociated virion control; the remaining dissociated virion samples were dialyzed overnight against 10%/ DMSO-0.01% Triton X-100 in phosphate-buffered saline (pH 7.4) with (B) 5 x i0-' M CaCl2, (C) 5 X 10-i; M CaCl2, and (D) 5 x 10-7 M CaCI2. Samples were then layered onto a 10 to 30%,o sucrose gradient and centrifuged in an SW50.1 rotor at 40,000 rpm for 40 min at 40C. Radioactivity determinations were made on the fractions, and the remainder was stored at 40C for further analysis. Arrow indicates sedimentation position of 240S polyoma virions centrifuged in a parallel tube. All samples were labeled with [3H]thymidine.
the gradient. However, when the dissociated virions were dialyzed against reassembly buffer containing various concentrations of Ca2", it was found that dissociated virions were reassembled to varying degrees and sedimented in the 240S region of the gradient. When dissociated polyoma virions were dialyzed against reassembly buffer containing 5 x 10-5 M Ca2", 17% of the radioactive counts in the gradient were found in the 240S region (Fig. 1B), whereas 5 x 10-6 M Ca2" allowed 22% (Fig. 1C) and 5 x 10-7 M Ca2" allowed 33% reassembly (Fig. 1D). Apparently, higher concentrations of Ca2" are inhibitory for polyoma reassembly. Complete removal of Ca2" from the reassembly buffer was also found to decrease reassembly of polyoma virions. Infectivity of the respective reassembled virion preparations will be described subsequently. Susceptibility of reassembled virions to DNase. After dissociation, a sample of the dissociated virion preparation was analyzed by velocity sedimentation in sucrose gradients before and after treatment with pancreatic DNase 1 (Fig. 2). Prior to DNase treatment, only one peak of radioactivity was found, corresponding to the 48S DNA-protein complex, in the dissociated virion preparation (Fig. 2A). When the dissociated virion preparation was subjected to DNase treatment before centrifugation, only soluble DNA fragments could be seen at the top of the gradient (Fig. 2B). After dialysis of the dissociated virion sample against the reassembly buffer, the samples were again analyzed by sedimentation velocity sucrose gradients before and after treatment with pancreatic DNase. Prior to DNase treatment, the reassembled virion sample was found to contain not only a large peak of radioactivity in the region of polyoma complete virions, but also, apparently, many species of intermediate particles which had not reached completion of reassembly (Fig. 2A). Particles with a sedimentation value of 48S (DNA-protein complex) to 240S (polyoma completes) were evident in the gradient profile. After DNase treatment of the reassembled virion preparation, only one peak of radioactivity, which sedimented at 240S, was detected in the gradient (Fig. 2B). From the radioactivity present in the region of 240S in Fig. 2A and B, it was estimated that 60% of the reassembled 240S species was resistant to DNase treatment. Conversely, virtually all of the intermediate reassembled products (fractions 15 to 30) were susceptible to DNase treatment. Buoyant density analysis of reassembled polyoma virions. To further examine the biophysical properties of the reassembled polyoma virions, fractions 11 to 15 (Fig. 2) from the sucrose gradients (240S region) were pooled and
643
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VOL. 32, 1979
region of reassembled polyoma virions (data not shown). The results of these experiments indicate that the particles sedimenting at 240S in the sucrose gradients were likely virions and not aggregates of DNA-protein complex and capsomere subunits which just fortuitously sedimented at 240S. In addition, the reassembled virions (with or without DNase treatment) would appear to be stable to CsCl. Little or no radioactivity was found in the pellet of the CsCl gradient, where free DNA or DNA-protein complex would be found. Electron microscopy of intact, dissociated, and reassembled polyoma virions. The reassembly of polyoma virions was also examined by electron microscopy. Polyoma virions before dissociation can be seen in Fig. 4A. After dissociation of the virions with EGTAME, the virions were reduced to free DNA-protein complexes and capsomere subunits (Fig. 4B). No intact virions or partially dissociated virions could be detected in the dissociated vir-
i0 cr
0
1.40
201 10
20
30
'
CJ)
40
1.30
T
B FRACTION NUMBER
FIG. 2. Velocity sedimentation of EGTA-ME-dissociated and reassembled polyoma virions after treatment with DNase 1. [3H]thymidine-labeledpolyoma virions were exposed to 1 mM EGTA-0.1 M ME-0.15 M NaCl in 0.05 M Tris buffer (pH 7.4) for 30 min at room temperature. The dissociated virion sample was then divided into two equal portions. The dissociated virion control sample was stored at 4°C. The remaining sample was dialyzed against 5 x 10-' M CaCl2-10% DMSO-0.01% Triton X-100 in phosphate-buffered saline (pH 7.4). A portion of the dissociated virion and reassembled virion preparations was treated with 50 yg ofpancreatic DNase 1 per ml 30 min at 37°C. Samples were layered onto 10 to 30%o sucrose gradients and centrifuged in an SW50.1 rotor at 40,000 rpm for 40 min at 4'C. Radioactivity determinations were made on the fractions, and the remainder was stored at 4°C for further analysis. (A) No DNase treatment; (B) DNase treated. (O EGTAME-dissociated polyoma virions; (0) reassembled polyoma virions.
analyzed by equilibrium CsCl centrifugation (Fig. 3). Control polyoma virions banded at a density of 1.315 g/cm3. The reassembled virions, with or without DNase treatment, were also found to have a density of 1.315 g/cm3. In similar experiments, in which fractions 11 to 15 were pooled from dissociated virion sucrose gradients, no radioactivity could be detected in the density
,N0 16
1.25
>_ Cl) z
x
w
1.20 a
'X- 12
1.15
0
I8 0
4
10
40 30 20 FRACTION NUMBER FIG. 3. Buoyant density determination ofpolyoma virions and reassembled polyoma virions in CsCl gradients. Polyoma virions and reassembled virions (with and without DNase I treatment) were isolated from sucrose gradients as shown in Fig. 2. Samples were layered onto preformed CsCl gradients and centrifuged in an SW50.1 rotor at 35,000 rpm for 20 h at 10°C. Fractions were collected and assayed for radioactivity and density determinations. Arrow indicates density of control polyoma virions. Symbols: (0) reassembled polyoma virions, no DNase treatment; (0) reassembled polyoma virions, DNase treated.
644
BRADY, KENDALL, AND CONSIGLI
J. VIROL.
IIP A
'1)r,VI)
..
'U.:i
-...:
-
C
D
1;
FIG. 4. Electron microscopy of intact, EGTA-ME-dissociated, and reassembled polyoma virions. (A) Control; intact polyoma virions; (B) EGTA-ME-dissociated polyoma virions; (C) reassembled polyoma virions; (D) reassembled polyoma virions isolated from 240S region of sucrose gradient as shown in Fig. 2. Samples were mounted on specimen grids, stained with uranyl acetate, and examined. x180,000.
ion preparations. Following dialysis of the dissociated virion preparation against the reassembly buffer, however, a mixture of virions, DNAprotein complexes, and capsomere subunits could be visualized in the electron microscope (Fig. 4C). As an additional control to insure that the particles shown in Fig. 4C were indeed intact virions, the sample was centrifuged on velocity sedimentation sucrose gradients as shown in Fig. 1, and the 240S region was isolated. Samples of the reassembled 240S particles were then examined by electron microscopy. Polyoma virions identical to those before dissociation were found in the isolated 240S fraction (Fig. 4D). Thus, if one compares the biophysical properties of intact, dissociated, and reassembled polyoma virions, the intact virions and reassembled virions are virtually identical, whereas the dissociated virions are markedly different (Table 1). The only significant difference between the intact and reassembled virions was the partial sensitivity of the reassembled virions to DNase treatment. Approximately 40% of the reassembled 240S virions were sensitive to DNase treat-
ment. However, the reassembled virions resistant to DNase treatment would appear to be quite "tight," since 93% of the DNA found inside these virions was found to be component I polyoma DNA (data not shown).
Analysis of hemagglutinating activity and plaque-forming ability of reassembled polyoma virions. Since the reassembled viri.ons appeared similar to intact virions by biophysical examination, the next series of experiments were performed to determine the biological properties of the reassembled virions. After the isolation of the 240S region from sucrose gradients containing intact virions, dissociated virions, and reassembled virions, the three samples were assayed for HA activity, infectivity by the immunofluorescent antibody assay, and plaque-forming ability (Table 2). When the three preparations were assayed for HA activity, the control untreated virions (amount of virus applied to gradient was equal to that added to dissociation mix) had an HA titer of 5,120 HA units per ml, whereas the reassembled virions had a titer of 640 HA units per ml. The dissoci-
REASSEMBLY OF INFECTIOUS POLYOMA VIRIONS
VOL. 32, 1979
645
TABLE 1. Comparison of biophysical properties of intact, dissociated, and reassembled polyoma virions % Compo% DNase Density Virion sample
Intact Dissociated
Reassembled
S2.
(sucrose)
240 48S complex; 18, 12, 5S capsomeres 240
TABLE 2. Comparison of biological properties of intact, dissociated, and reassembled polyoma virions PFU/ml HA titer FAM Virion sample (+) 3 x 108 5,120 Intact 0 (-)
