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Parameters affecting the stability of SV40 virions during the extraction of nucleoprotein complexes
VIROLOGY
95,
256-259 (1979)
Parameters Affecting the Extraction
MICHAEL Princeton
SEIDMAN, University,
the Stability of SV40 Virions during of Nucleoprotein Complexes
ELLEN
Department
GARBER,
of Biochemical Accepted
February
AND ARNOLD
Sciences, Princeton,
J. LEVINE’ New
Jersey
08540
12, 1979
The extraction of SV40 nucleoprotein complexes from infected cell nuclei frequently results in the disruption of mature SV40 virions which then contaminate the nucleoprotein complexes (E. Garber, M. Seidman, and A. J. Levine, Virology, 90, 305-316, 1978). The parameters that are important in maintaining SV40 virion integrity during this extraction procedure have been studied. The most important factors affecting virion stability in nuclear extracts are: (1) the ionic strength of the solution should be between 50 and 100 n&f KCl, (2) chelating agents must be omitted from the nuclear extracts but can be employed in subsequent analyses, and (3) cellular factors in the nuclear extract can affect virion stability and the concentration of nuclei should be kept below 10’ nuclei/ml of extraction buffer. Other components of the extraction buffer such as pH and protease inhibitors appear to be less important for the integrity of SV40 vii-ions.
In SV40-infected monkey cells the form I viral DNA is associated with cellular histones and virion structural proteins. Several different procedures have been developed which extract these SV40 nuclear protein complexes from the infected cells or nuclei. The size, shape, and proteins associated with these complexes vary depending upon the extraction conditions employed. In moderately high ionic strength solutions (400 mM) viral DNA is associated with histones and VP-l, sediments at 55 S (1-3) and has a conformation of histone octomer beads on a string. In lower ionic strength buffers this complex is more compact and sediments at ‘75-85 S (4,5). Recently it has been shown that the great majority of these nucleoprotein complexes described previously resulted from the breakdown of 250 S virions in the extraction buffers and as such these complexes did not accurately reflect the in viva situation (6). In order to proceed with an analysis of the natural in viva nucleoprotein complexes, it is important to define the variables which affect the stability of SV40 virions in nuclear extracts so that virion degradation products do not con1 To whom reprint requests should be sent. 0042~6822/79/070256-04$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
256
taminate the viral complexes under study. This communication elucidates some of the factors which influence virion stability during extraction of SV40 nucleoprotein complexes. The most important parameters affecting virion stability in nuclear extracts appear to be: (1) the ionic strength of the solution must be between 50 and 100 n-U KCl, (2) chelating agents must be omitted from the nuclear extracts, and (3) cellular factors in the nuclear extract can affect virion stability and so the concentration of nuclei should be kept below 10’ nuclei/ml of extraction buffer. Other parameters such as pH (6.5-8.0) and the presence or absence of protease inhibitors appear to be less important. To characterize the parameters that were important for SV40 virion stability during extraction of viral nucleoprotein complexes the following experiments were performed. Monolayer cultures of BSC-1 cells were infected with SV40 as described previously (6). At 30 hr after infection the cells were labeled with [3H]thymidine (1-5 @.X/ml) for 14 hr. The cells were harvested at 44 hr after infection and nuclei were prepared (6). These nuclei were suspended in either 10 mM Pipes buffer at pH 6.8 or 10 mM
257
SHORT COMMUNICATIONS
HEPES buffer pH 8.0 containing 1% Triton X-100, 0.5% Brij 58, and 0.3 mg/ml phenylmethylsulfonyfluoride and 100 units/ml Trasylol. In different experiments the ionic strength of the extraction buffer was varied by addition of KCl. In some experiments either 1 mM EDTA or 0.5 mM MgCl, was added. After a 1-hr extraction of the nuclei (10’ nuclei/ml) in these different solutions at O-4” with occasional mixing, the nuclei were centrifuged out of solution and the soluble complexes were analyzed by sedimentation through 5-30% sucrose gradients containing 10 mM Pipes, pH 6.8, 100 mM KCI, and 1 n&f EDTA. Figure 1 presents the sedimentation profiles of [3H]thymidine-labeled SV40 nucleoprotein complexes obtained from four different extraction procedures. When SV40
18 16t
A
0
1
R
TUBE
nucleoprotein complexes were extracted in 10 mM Pipes, pH 6.8 (or 10 mM HEPES, pH 8.0), 100 mM KCl, 1% Triton X-100, and 0.5% Brij 58 the great majority of the viral DNA labeled for 14 hr with [3H]thymidine sedimented at 250 S, in the position expected for mature virions (Fig. lC> (6). Lowering the ionic strength to 5 mM KC1 during the extraction procedure (but not in the sucrose gradient) resulted in an extensive disruption of the 250 S virion component (Fig. 1A). The addition of 0.5 mM MgCl, partially (about 30-40%) protected some of the nucleoprotein complex sedimenting at 250 S (Fig. lB), however, this 250 S material appears to be unstable in the high ionic strength CsCl solutions used for virion purification (7) and as such may not be equivalent to well-characterized SV40 virions. By employing 400 n-J4 KC1 in the extraction buffer, there was complete breakdown of the 250 S virion (Fig. 1D). Only the 100 mM KC1 conditions produced a 250 S virion that was also stable in CsCl solutions used to purify the virus (6). Figure 2 shows a compilation of these results and many other experiments of this type. These data are plotted as the percentage of 250 S virion component isolated (% of [3H]thymidine-labeled cpm sedimenting at 250 S) as a function of the ionic strength of the extraction buffer. Maximum amounts of stable virus were obtained between 50 and 100 mM KCl. Lower or higher ionic strength solutions lead to virion instability. The addition of 1 mM EDTA, even when
NUMBER
FIG. 1. SV40-Infected BSC-1 cells were labeled from 26 to 40 hr PI with [3H]thymidine. Nuclei were prepared as described by Garber et al. (6). The nuclei were incubated in the appropriate extraction buffers for 1 hr at 0”. The nuclei were removed by centrifugation and the extracts were centrifuged on sucrose gradients in 100 m&f KC1 as described (6). The extraction buffers contained 10 n&f HEPES, pH 8.0, 1% Triton X-100, 0.5% Brij 58, PMSF, Trasylol, and (A) 5 mM KCI, (B) 5 m&f KCl, 0.5 mM MgCl*, (C) 100 mkf KCI, or (D) 400 r&f KCl.
,Oii
/
5
50
”
100
n
150
y-:----%&
200 iKCI1
250
3ho--i50
400
FIG. 2. The protocol described in Fig. 1 was employed. The percentage of 13H]thymidine counts per minute in the gradients which appeared in the 250 S peak was determined. Symbols: ionic strength (mM KCl), 0; 1 mdf EDTA added to extraction buffer, 0; 0.5 mM MgCIZ, 0.
253
SHORT COMMUNICATIONS
100 n-&f KC1 was employed in the extraction solution, resulted in the breakdown of SV40 virions (Fig. 2). Consistent with this observation is the fact that 0.5 mM MgCl, added to solutions of 5 mM KC1 extraction buffer, partially protected nucleoprotein complexes that sedimented like virions (250 S) (Fig. 2). These complexes however were not stable in CsCl solutions used to purify virus (7). At ionic strengths between 50 and 100 mM KCl, the buffer (Pipes or HEPES) and pH (6.8-8.0) did not alter the stability of SV40 virions extracted from nuclei. The addition of PMSF or Trasylol protease inhibitors had little effect upon the sedimentation properties of SV40 nucleoprotein complexes. The presence or absence of 1 mM EDTA in the sucrose gradient solutions employed to analyze SV40 nucleoprotein complexes and virions did not affect the sedimentation properties of these complexes. EDTA (1 n&I) was employed in the sucrose gradients when the purpose of the experiment was to study viral DNA from SV40 nucleoprotein complexes (75 S chromosome). These complexes are suseptible to nuclease attack (8) and the chelators minimize this problem. As a second criteria for SV40 virion stability the [3H]thymidine-labeled material from the 200-250 S region of these sucrose gradients (Fig. 1) were centrifuged to equilibrium in CsCl gradients to determine its density and stability to high ionic strength solutions. This region of the sucrose gradient has been shown to contain two distinct forms of SV40 nucleoprotein complexes, a 200 S precursor to mature virions, the majority of which is unstable in C&l, and the salt stable 250 S virions (6). Thus the appearance of mature SV40 virions can be detected with time of labeling by assaying for a 250 S sedimenting component with a density of 1.33 g/ml in CsCI. ,The remainder of the [3H]thymidine counts per minute that sediment in the 200-250 S region of the sucrose gradient can be attributed to the 200 S previrion component or defective viruses. To carry out this analysis SV40-infected BSC-1 cells were labeled with [3H]thymidine for either 1,2,4, or 16 hr and harvested at 40 hr after
infection. Nuclei from these cells were extracted with 10 n&! Pipes, pH 6.8, 1% Triton X-100,0.5% Brij 58,100 mM KCl, 0.3 mg/ml PMSF, and 100 units/ml Trasylol. The soluble nuclear extracts were then sedimented through a 5-30% sucrose gradient in 10 mM Pipes, pH 6.8, and 100 mM KCl. The 3H-labeled components that sedimented between 200 and 250 S in the sucrose gradient were then centrifuged to equilibrium in CsCl. Because CaCl, has been implicated as an important factor in papovavirus stability (9) 1 m&I CaCl, was added to one-half of each sample prior to centrifugation in CsCl to assess the importance of this parameter. The results of this experiment are presented in Table 1 and are expressed as the percentage of [3H]thymidine counts per minute (from the 200-250 S region of a sucrose gradient) that are recovered at the density of SV40 virions, 1.33 g/ml, as a function of labeling times. After a 1-hr labeling time about 20% of the rapidly sedimenting components are in saltstable virions. This percentage rises, as virions accumulate, over a 4-hr period of labeling and approaches a maximum level seen in the 14-hr labeling of infected cells. When [3H]leucine was employed to label SV40 virions (in the 200-250 S region of a TABLE
1
CESIUM CHLORIDE STABILITY OF 200-250 S MATERIAL AS A FUNCTION OF LABELING TIMES Percentage of 3H-cpm found in 1.33 g/ml peak in CsCl gradients Time (hr)
Isotope
No CaCl,
1 mM CaCl,
1 2 4 16 16
[3H]TdR [3H]TdR [3H]TdR [3H]TdR [3H]leu
21 38 55 6’7 69
23 41 61 75 71
” Fast-sedimenting complexes were centrifuged to equilibrium in CsCl, and the percentage of 3H-cpm found in the 1.33 g/ml virion peak was determined. [3H]Tdr is [3H]thymidine, [3H]leu is [3H]leucine, employed to label the virus.
SHORT
COMMUNICATIONS
sucrose gradient) identical results were obtained (Table 1). It is not clear why only 70-75% of the material sedimenting at 200250 S had a density of 1.33 g/ml in CsCl even with 16-hr labeling times. The addition of CaCl, had only a small effect upon virion stability as tested by this protocol. However, if the 250 S virions are to be stored for periods of time greater than 24 hr (especially if EDTA is present) then the addition of CaCl, is essential for virion stability in CsCl solutions. The experiments presented here elucidate some of the important variables for SV40 virion stability during extraction of viral nucleoprotein complexes from infected cell nuclei. These major parameters are (1) the ionic strength of the extraction solution which should be between 50 and 100 mM KCl, (2) chelators such as EDTA must be eliminated from the extraction solution but may be used in subsequent analyses, and (3) cellular factors in the nuclear extract play a role in virion disruption (6) and so the concentration of nuclei should be kept below 10’ nuclei/ml of extraction buffer. In addition, the use of different monkey cell lines (BSC-1, BSC-l,,, CV-1, or primary AGMK) may have an effect upon the proportions,
259
obtained in any experiment, of 250 S virion and its breakdown products. One of the major products of virion disruption in vitro appears to be the 75 S SV40 minichromosome (Fig. 1D) (6). It is possible that the instability of SV40 virions in crude nuclear extracts in vitro may be analogous to the virus uncoating reactions which permit virus infection to begin in vivo. REFERENCES 1. GREEN, M. J., MILLER, H. I., and HENDLER, S., Proc. Nut. Acad. Sci. USA 68,1032-X)36(1971). 2. WHITE, M., and EASON, R., J. Viral. 8, 363-371 (1971). 3. SEN, A., HANCOCK, R., and LEVINE, A. J., Virology 61, 11-21 (1974). 4. VARSHAVSKY, A. J., BAKAYEV, V., CHUMACKOV, P. M., and GEORGIEV, G. P., Nucl. Acids Res. 3, 2101-2113 (1976). 5. CHRISTIANSEN, G., and GRIFFITH, J., Nucl. Acids Res. 4, 1837-1881 (1977). 6. GARBER, E., SEIDMAN, M., and LEVINE, A. J., Virology 90, 305-316 (1978). 7. BAUMGARTNER, I., KUHN, C., and FANNING, E., Virology, in press (1979). 8. SCOTT, W. A., and WIGMORE, D., Cell 15, 15111518 (1978). 9. BRADY, J. N., WINSTON, V. D., and CONSIGLI, R. A., J. Viral. 23, 717-724 (1977).
95,
256-259 (1979)
Parameters Affecting the Extraction
MICHAEL Princeton
SEIDMAN, University,
the Stability of SV40 Virions during of Nucleoprotein Complexes
ELLEN
Department
GARBER,
of Biochemical Accepted
February
AND ARNOLD
Sciences, Princeton,
J. LEVINE’ New
Jersey
08540
12, 1979
The extraction of SV40 nucleoprotein complexes from infected cell nuclei frequently results in the disruption of mature SV40 virions which then contaminate the nucleoprotein complexes (E. Garber, M. Seidman, and A. J. Levine, Virology, 90, 305-316, 1978). The parameters that are important in maintaining SV40 virion integrity during this extraction procedure have been studied. The most important factors affecting virion stability in nuclear extracts are: (1) the ionic strength of the solution should be between 50 and 100 n&f KCl, (2) chelating agents must be omitted from the nuclear extracts but can be employed in subsequent analyses, and (3) cellular factors in the nuclear extract can affect virion stability and the concentration of nuclei should be kept below 10’ nuclei/ml of extraction buffer. Other components of the extraction buffer such as pH and protease inhibitors appear to be less important for the integrity of SV40 vii-ions.
In SV40-infected monkey cells the form I viral DNA is associated with cellular histones and virion structural proteins. Several different procedures have been developed which extract these SV40 nuclear protein complexes from the infected cells or nuclei. The size, shape, and proteins associated with these complexes vary depending upon the extraction conditions employed. In moderately high ionic strength solutions (400 mM) viral DNA is associated with histones and VP-l, sediments at 55 S (1-3) and has a conformation of histone octomer beads on a string. In lower ionic strength buffers this complex is more compact and sediments at ‘75-85 S (4,5). Recently it has been shown that the great majority of these nucleoprotein complexes described previously resulted from the breakdown of 250 S virions in the extraction buffers and as such these complexes did not accurately reflect the in viva situation (6). In order to proceed with an analysis of the natural in viva nucleoprotein complexes, it is important to define the variables which affect the stability of SV40 virions in nuclear extracts so that virion degradation products do not con1 To whom reprint requests should be sent. 0042~6822/79/070256-04$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
256
taminate the viral complexes under study. This communication elucidates some of the factors which influence virion stability during extraction of SV40 nucleoprotein complexes. The most important parameters affecting virion stability in nuclear extracts appear to be: (1) the ionic strength of the solution must be between 50 and 100 n-U KCl, (2) chelating agents must be omitted from the nuclear extracts, and (3) cellular factors in the nuclear extract can affect virion stability and so the concentration of nuclei should be kept below 10’ nuclei/ml of extraction buffer. Other parameters such as pH (6.5-8.0) and the presence or absence of protease inhibitors appear to be less important. To characterize the parameters that were important for SV40 virion stability during extraction of viral nucleoprotein complexes the following experiments were performed. Monolayer cultures of BSC-1 cells were infected with SV40 as described previously (6). At 30 hr after infection the cells were labeled with [3H]thymidine (1-5 @.X/ml) for 14 hr. The cells were harvested at 44 hr after infection and nuclei were prepared (6). These nuclei were suspended in either 10 mM Pipes buffer at pH 6.8 or 10 mM
257
SHORT COMMUNICATIONS
HEPES buffer pH 8.0 containing 1% Triton X-100, 0.5% Brij 58, and 0.3 mg/ml phenylmethylsulfonyfluoride and 100 units/ml Trasylol. In different experiments the ionic strength of the extraction buffer was varied by addition of KCl. In some experiments either 1 mM EDTA or 0.5 mM MgCl, was added. After a 1-hr extraction of the nuclei (10’ nuclei/ml) in these different solutions at O-4” with occasional mixing, the nuclei were centrifuged out of solution and the soluble complexes were analyzed by sedimentation through 5-30% sucrose gradients containing 10 mM Pipes, pH 6.8, 100 mM KCI, and 1 n&f EDTA. Figure 1 presents the sedimentation profiles of [3H]thymidine-labeled SV40 nucleoprotein complexes obtained from four different extraction procedures. When SV40
18 16t
A
0
1
R
TUBE
nucleoprotein complexes were extracted in 10 mM Pipes, pH 6.8 (or 10 mM HEPES, pH 8.0), 100 mM KCl, 1% Triton X-100, and 0.5% Brij 58 the great majority of the viral DNA labeled for 14 hr with [3H]thymidine sedimented at 250 S, in the position expected for mature virions (Fig. lC> (6). Lowering the ionic strength to 5 mM KC1 during the extraction procedure (but not in the sucrose gradient) resulted in an extensive disruption of the 250 S virion component (Fig. 1A). The addition of 0.5 mM MgCl, partially (about 30-40%) protected some of the nucleoprotein complex sedimenting at 250 S (Fig. lB), however, this 250 S material appears to be unstable in the high ionic strength CsCl solutions used for virion purification (7) and as such may not be equivalent to well-characterized SV40 virions. By employing 400 n-J4 KC1 in the extraction buffer, there was complete breakdown of the 250 S virion (Fig. 1D). Only the 100 mM KC1 conditions produced a 250 S virion that was also stable in CsCl solutions used to purify the virus (6). Figure 2 shows a compilation of these results and many other experiments of this type. These data are plotted as the percentage of 250 S virion component isolated (% of [3H]thymidine-labeled cpm sedimenting at 250 S) as a function of the ionic strength of the extraction buffer. Maximum amounts of stable virus were obtained between 50 and 100 mM KCl. Lower or higher ionic strength solutions lead to virion instability. The addition of 1 mM EDTA, even when
NUMBER
FIG. 1. SV40-Infected BSC-1 cells were labeled from 26 to 40 hr PI with [3H]thymidine. Nuclei were prepared as described by Garber et al. (6). The nuclei were incubated in the appropriate extraction buffers for 1 hr at 0”. The nuclei were removed by centrifugation and the extracts were centrifuged on sucrose gradients in 100 m&f KC1 as described (6). The extraction buffers contained 10 n&f HEPES, pH 8.0, 1% Triton X-100, 0.5% Brij 58, PMSF, Trasylol, and (A) 5 mM KCI, (B) 5 m&f KCl, 0.5 mM MgCl*, (C) 100 mkf KCI, or (D) 400 r&f KCl.
,Oii
/
5
50
”
100
n
150
y-:----%&
200 iKCI1
250
3ho--i50
400
FIG. 2. The protocol described in Fig. 1 was employed. The percentage of 13H]thymidine counts per minute in the gradients which appeared in the 250 S peak was determined. Symbols: ionic strength (mM KCl), 0; 1 mdf EDTA added to extraction buffer, 0; 0.5 mM MgCIZ, 0.
253
SHORT COMMUNICATIONS
100 n-&f KC1 was employed in the extraction solution, resulted in the breakdown of SV40 virions (Fig. 2). Consistent with this observation is the fact that 0.5 mM MgCl, added to solutions of 5 mM KC1 extraction buffer, partially protected nucleoprotein complexes that sedimented like virions (250 S) (Fig. 2). These complexes however were not stable in CsCl solutions used to purify virus (7). At ionic strengths between 50 and 100 mM KCl, the buffer (Pipes or HEPES) and pH (6.8-8.0) did not alter the stability of SV40 virions extracted from nuclei. The addition of PMSF or Trasylol protease inhibitors had little effect upon the sedimentation properties of SV40 nucleoprotein complexes. The presence or absence of 1 mM EDTA in the sucrose gradient solutions employed to analyze SV40 nucleoprotein complexes and virions did not affect the sedimentation properties of these complexes. EDTA (1 n&I) was employed in the sucrose gradients when the purpose of the experiment was to study viral DNA from SV40 nucleoprotein complexes (75 S chromosome). These complexes are suseptible to nuclease attack (8) and the chelators minimize this problem. As a second criteria for SV40 virion stability the [3H]thymidine-labeled material from the 200-250 S region of these sucrose gradients (Fig. 1) were centrifuged to equilibrium in CsCl gradients to determine its density and stability to high ionic strength solutions. This region of the sucrose gradient has been shown to contain two distinct forms of SV40 nucleoprotein complexes, a 200 S precursor to mature virions, the majority of which is unstable in C&l, and the salt stable 250 S virions (6). Thus the appearance of mature SV40 virions can be detected with time of labeling by assaying for a 250 S sedimenting component with a density of 1.33 g/ml in CsCI. ,The remainder of the [3H]thymidine counts per minute that sediment in the 200-250 S region of the sucrose gradient can be attributed to the 200 S previrion component or defective viruses. To carry out this analysis SV40-infected BSC-1 cells were labeled with [3H]thymidine for either 1,2,4, or 16 hr and harvested at 40 hr after
infection. Nuclei from these cells were extracted with 10 n&! Pipes, pH 6.8, 1% Triton X-100,0.5% Brij 58,100 mM KCl, 0.3 mg/ml PMSF, and 100 units/ml Trasylol. The soluble nuclear extracts were then sedimented through a 5-30% sucrose gradient in 10 mM Pipes, pH 6.8, and 100 mM KCl. The 3H-labeled components that sedimented between 200 and 250 S in the sucrose gradient were then centrifuged to equilibrium in CsCl. Because CaCl, has been implicated as an important factor in papovavirus stability (9) 1 m&I CaCl, was added to one-half of each sample prior to centrifugation in CsCl to assess the importance of this parameter. The results of this experiment are presented in Table 1 and are expressed as the percentage of [3H]thymidine counts per minute (from the 200-250 S region of a sucrose gradient) that are recovered at the density of SV40 virions, 1.33 g/ml, as a function of labeling times. After a 1-hr labeling time about 20% of the rapidly sedimenting components are in saltstable virions. This percentage rises, as virions accumulate, over a 4-hr period of labeling and approaches a maximum level seen in the 14-hr labeling of infected cells. When [3H]leucine was employed to label SV40 virions (in the 200-250 S region of a TABLE
1
CESIUM CHLORIDE STABILITY OF 200-250 S MATERIAL AS A FUNCTION OF LABELING TIMES Percentage of 3H-cpm found in 1.33 g/ml peak in CsCl gradients Time (hr)
Isotope
No CaCl,
1 mM CaCl,
1 2 4 16 16
[3H]TdR [3H]TdR [3H]TdR [3H]TdR [3H]leu
21 38 55 6’7 69
23 41 61 75 71
” Fast-sedimenting complexes were centrifuged to equilibrium in CsCl, and the percentage of 3H-cpm found in the 1.33 g/ml virion peak was determined. [3H]Tdr is [3H]thymidine, [3H]leu is [3H]leucine, employed to label the virus.
SHORT
COMMUNICATIONS
sucrose gradient) identical results were obtained (Table 1). It is not clear why only 70-75% of the material sedimenting at 200250 S had a density of 1.33 g/ml in CsCl even with 16-hr labeling times. The addition of CaCl, had only a small effect upon virion stability as tested by this protocol. However, if the 250 S virions are to be stored for periods of time greater than 24 hr (especially if EDTA is present) then the addition of CaCl, is essential for virion stability in CsCl solutions. The experiments presented here elucidate some of the important variables for SV40 virion stability during extraction of viral nucleoprotein complexes from infected cell nuclei. These major parameters are (1) the ionic strength of the extraction solution which should be between 50 and 100 mM KCl, (2) chelators such as EDTA must be eliminated from the extraction solution but may be used in subsequent analyses, and (3) cellular factors in the nuclear extract play a role in virion disruption (6) and so the concentration of nuclei should be kept below 10’ nuclei/ml of extraction buffer. In addition, the use of different monkey cell lines (BSC-1, BSC-l,,, CV-1, or primary AGMK) may have an effect upon the proportions,
259
obtained in any experiment, of 250 S virion and its breakdown products. One of the major products of virion disruption in vitro appears to be the 75 S SV40 minichromosome (Fig. 1D) (6). It is possible that the instability of SV40 virions in crude nuclear extracts in vitro may be analogous to the virus uncoating reactions which permit virus infection to begin in vivo. REFERENCES 1. GREEN, M. J., MILLER, H. I., and HENDLER, S., Proc. Nut. Acad. Sci. USA 68,1032-X)36(1971). 2. WHITE, M., and EASON, R., J. Viral. 8, 363-371 (1971). 3. SEN, A., HANCOCK, R., and LEVINE, A. J., Virology 61, 11-21 (1974). 4. VARSHAVSKY, A. J., BAKAYEV, V., CHUMACKOV, P. M., and GEORGIEV, G. P., Nucl. Acids Res. 3, 2101-2113 (1976). 5. CHRISTIANSEN, G., and GRIFFITH, J., Nucl. Acids Res. 4, 1837-1881 (1977). 6. GARBER, E., SEIDMAN, M., and LEVINE, A. J., Virology 90, 305-316 (1978). 7. BAUMGARTNER, I., KUHN, C., and FANNING, E., Virology, in press (1979). 8. SCOTT, W. A., and WIGMORE, D., Cell 15, 15111518 (1978). 9. BRADY, J. N., WINSTON, V. D., and CONSIGLI, R. A., J. Viral. 23, 717-724 (1977).