VIROLOGY
94, 219-223
(197%
Early Events in the Infection of Permissive Comparison of Chymotrypsin-Treated
VERA CHLUMECKA, Department
of Biochemistry,
Cells with Polyoma Virus: and Untreated Virus
PERRY D’OBRENAN, University
of Alberta,
AND
Edmonton,
JOHN S. COLTERl
Alberta
T6G 2H7, Canada
Accepted December 5, 1978 An examination of the early events in the infection of cultured mouse embryo fibroblasts using both chymotrypsin-treated (chymo+) and untreated (chymo-) polyoma virus has shown that exposure to the protease has no effect on the ability of the virions to attach to, penetrate into, and enter the nuclei of cells. Uncoating, which our data indicate takes place exclusively in the nuclei, appears to be somewhat delayed in the case of the chymo+ virions. The only identifiable product of uncoating is a DNA protein complex having a sedimentation coefficient of the order of 52-55 S. Stimulation of cellular and viral DNA synthesis was found to be greatly reduced in cells infected with chymo+ virions relative to those infected with chvmo- virions. but the specific infectivities of DNAs isolated from chymo+ and chymovirions were found to be pre&ly the same.
It was reported earlier (2) that incubation of polyoma (Py) virus with chymotrypsin during purification results in a marked decrease in the recovery of infectious virus (PFU) but not of hemagglutinating activity (HAU), and that exposure to the protease has no effect on the physical integrity of either the virion or its DNA but leads to the appearance in the virions of a new polypeptide, apparently produced from VP3 by chymotryptic cleavage. We report here the results of an investigation in which Py virus exposed to chymotrypsin during purification (chymo+ virions) and Py virus purified without exposure to the protease (chymo- virions) were examined with respect to the early events of the infectious cycle. In addition, the abilities of chymo’ and chymo- virions to stimulate the synthesis of cellular and viral DNA were compared, as were the infectivities of DNAs isolated from the two preparations. Tertiary cultures of mouse embryo (ME) nbroblasts, used in all experiments described herein, were established and grown as described previously (1). Virus pools were prepared by infecting confluent mouse kidney cultures at a m.o.i. of 1 and harvesting when cell lysis was complete. To prepare labeled ’ To whom reprint
requests
should be sent. 219
virus, the medium on the infected cultures was suppIemented with [3H]thymidine (20 FCi/ml). The virus was purified as described previously (1). Virus pools were carried through the first three steps of the purification procedure (methanol precipitation, sequential treatment with receptor destroying enzyme (RDE), and RNase + DNase) and then divided into two equal aliquots, only one of which was incubated with a-chymotrypsin before the purification was completed. This led in each case to the production of two preparations (chymo+ and chymo-) that contained the same concentration of HAUs and physical particles, and, when the vii-ions were labeled, the same specific activity (counts per minute per physical particle). DNAs, isolated by phenol extraction from equal aliquots of chymo+ and chymo- virions, had infectivities [measured in ME monolayers using the DEAE-dextran procedure @)] of 1.4 x lo6 and 1.2 x lo6 PFU/ ml, respectively. Both contained 5 pg DNA/ ml. The corresponding virus suspensions had titers of 4.2 x 10s and 4.9 x lOlo PFU/ml. The synthesis of cellular and viral DNAs in cells infected with chymo+ and chymo0042~6822/79/050219-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
220
SHORT COMMUNICATIONS
FIG. 1. (Upper) Kinetics of attachment of labeled chymo- and chymo+ Py virions to ME cells at 22”. At each time, the amount of radioactivity that had become firmly cell associated was measured and expressed as a percentage of the input radioactivity. (0) chymovirions; (0) chymo+ virions. (Lower) Kinetics of penetration of labeled chymo- and chymo+ Py virions into ME cells. Cultures to which virus had been attached at 2” and then incubated at 37” were treated with RDE at various times postattachment. The amount of radioactivity not removed by RDE was expressed as a percentage of the radioactivity that was cell associated at zero time. (0) chymo- virions; (0) chymo+ virions.
virions at equal input multiplicities of physical particles (50 PFU/cell of chymo- virions) was examined by pulse labeling infected monolayer-s for 30 min with [14C]thymidine (2 &i/ml) at various times from 0 to 36 hr postattachment, fractionating the pulse-labeled monolayers by the Hirt procedure (3), and monitoring cellular and viral DNA synthesis by measuring the acid-insoluble radioactivity in the Hirt pellet and supernatant, respectively. It was found (data not shown) that the onset of synthesis was delayed (by lo-12 hr) and the extent of synthesis of both species was much depressed in cells infected with chymo+ virions.
To study attachment, replicate monolayers of ME cells were infected with labeled ehymo+ and chymo- virions at equal input multiplicities of physical particles (50 PFUl cell of chymo- virions) and incubated at 22”. The amount of radioactivity that had become firmly cell associated (not removed by washing with PBS) was determined at various times and expressed as a percentage of the input radioactivity (5 x lo4 cpmlplate in both series). To examine penetration, monolayers were incubated with labeled virions for 2 hr at 2”, after which they were washed and incubated in DME -2% fetal calf serum (FCS) at 37”. Periodically thereafter, cultures were treated with RDE (‘750units in 3 ml phosphate-buffered saline (PBS), 15min at 37”) to remove virus still attached to the plasma membrane. The extent of penetration was estimated from measurements of the fraction of the radioactivity that remained cell associated after RDE treatment. The results of these studies are illustrated in Fig. 1. The kinetics of attachment of chymo+ and chymo- virions to cells were found to be essentially identical (attachment reaches a plateau in 2-3 hr with 60-70% of the virions firmly cell associated) as were the rates at which chymo+ and chymo- virions, attached to cells at 2”, penetrate into the cells during a subsequent incubation at 37”. The initial rate of penetration was found to be quite rapid (about 50% within 1 hr), after which virus continues to enter the cells at a decreased rate until, by 6-8 hr, 85-90% of the cell-associated virions can no longer be removed by RDE. The intracellular distribution of virus as a function of time post-attachment was studied as follows. Monolayers were infected by incubation with labeled virus for 2 hr at 2”, after which they were washed with PBS and incubated at 37” in DME-2% FCS. At intervals thereafter, duplicate cultures were washed with PBS and incubated with RDE (see above), after which they were washed twice with NTM buffer (0.14 M NaCl, 1.5 mk’ MgCl,, 10 mM Tris, pH 7.4) and lysed by incubation for 10 min at 0” in the samebuffer containing 0.5% NP-40. After removal of the cytoplasmic fraction, the nuclei were
221
SHORT COMMUNICATIONS
washed with and collected into NTM buffer and disrupted by sonication. The amount of acid-insoluble radioactivity in each fraction was measured before and after incubation with DNase and expressed as a percentage of the total cell associated radioactivity at time zero (i.e., after attachment at 2’). Illustrative data are presented in Fig. 2. Several things are worth noting. The patterns of intracellular distribution obtained with the chymo+ and chymo- preparations are strikingly similar, with the rates of nuclear entry of the two being essentially identical. We find no evidence of any uncoating in the cytoplasm, using susceptibility of the viral genome to DNase as the criteria, and, during the first 6-8 hr postattachment, less of the nuclear radioactivity appears to be DNase-sensitive in chymo+ virion-infected cells than in chymo- virioninfected cells.
0
2
4 6 8 IO 12 INCUBATION TIME (hrs)
To obtain information regarding the fate of those v&ions that enter the nuclei, cells were infected with labeled virus, incubated, treated with RDE, and separated into cytoplasmic and nuclear fractions as outlined above. The nuclei were then treated briefly (5-10 see) with a mixture of sodium deoxycholate and Triton X-100 to remove outer nuclear membranes and cytoplasmic contamination (4) before being solubilized in 0.5 A4 NaCl, 0.05 M MgC&, 0.01 M Tris, pH 7.4. The preparations were homogenized briefly by hand (five strokes, Dounce homogenizer) before being analyzed by velocity sedimentation in linear lo-40% sucrose density gradients. The radioactivity was found to be resolved into (1) a rapidly sedimenting peak, (2) intact virions, and (3) a peak of more slowly sedimenting material with a sedimentation coefficient of the order of 52-55 S (see Fig. 2). When solubilized
14
16
IO 20 FRACTION
30 40 NUMBER
FIG. 2. (Left) Intracellular distribution of labeled chymo- (panel A) and chymo+ (panel B) Py vii-ions in ME cells. At various times postattachment, cultures were treated with RDE and separated into cytoplasmic and nuclear fractions. The amount of acid-insoluble radioactivity in each was measured before and after DNase treatment, and expressed as a percentage of the total cell-associated radioactivity at time zero. Circles, cytoplasmic counts per minute; squares, nuclear counts per minute; closed symbols, before DNase; open symbols, after DNase. (Right) Centrifugal analysis of solubilized nuclei isolated at 14 hr postattachment from ME cells infected with labeled chymo- (panel A) and chymo+ (panel B) Py virions. Centrifugation was for 90 min at 35,000rpm (SW41 rotor, Beckman L5-65 centrifuge) through lo-40% linear sucrose gradients. Sedimentation was from right to left. The arrows indicate the position of markers, Py virions (l), Mengo virions (2), R17 virions (3), and Py DNA I (4) in parallel gradients.
SHORT COMMUNICATIONS
222
TABLE 1
DISTRIBUTIONOFRADIOACTIVITYINSOLUBILIZEDNUCLEI ISOLATEDFROM CELLSINFECTED WITH[~H]THYMIDINE-LABELED CHYMO+ANDCHYMO-POLYOMAVIRION~ Chymo- Pyvirus Hours postattachment
Fastsedimenting component
Virion peak
2 6 10 14 24
41.2 41.5 35.0 33.6 39.0
52.1 41.3 35.0 33.9 26.4
Chymo+ Py virus
material
Fastsedimenting component
Virion peak
material
6.7 17.2 30.0 32.5 34.6
54.6 48.7 39.8 39.6 34.4
45.4 42.5 47.4 38.6 34.0
0.0 8.8 12.8 21.8 31.6
52-55 S
52-55 S
a Nuclei were isolated and solubilized, and the distribution of radioactivity therein was determined by centrifugal analysis as outlined in the legend to Fig. 2. The amount of radioactivity in each peak is expressed as a percentage of the total radioactivity recovered in the three peaks.
nuclei were incubated with DNase prior to centrifugation, a small amount of radioactivity was lost from the fast-sedimenting peak, the virion peak was unaffected, and the 5255 S peak was eliminated completely. The results of sedimentation analyses of solubilized nuclei isolated from chymo+ and chymo- virion-infected cells at various times from 2 to 24 hr postattachment are summarized in Table 1. The 52-55 S material can be detected in cells infected with chymovirions as early as 2 hr postattachment and increases progressively in amount during the 24-hr period. The appearance of this material was found to be delayed by 4-6 hr in cells infected with chymo+ virions, although by 24 hr postattachment it reaches levels comparable to those found in cells infected with chymo- virions. The data presented here show that exposure of Py virus to chymotrypsin during purification has no significant effect on the ability of the virions to attach to, penetrate into, and enter the nuclei of permissive cells. Exposure to the protease seems to decrease the efficiency with which the treated virions are uncoated, but this difference in the behavior of chymo+ and chymo- virions is transitory and does not provide a satisfactory explanation for the approximately 2 log loss in infectivity resulting from exposure to chymotrypsin. Our conclusion that the
uncoating of Py virus takes place exclusively in the nuclei is at variance with that of Frost and Bourgaux (5) but in agreement with that of Mackay and Consigli (6) and of other investigators (7, 8) who have examined the uncoating of SV40. The only product of uncoating that we have found is the material sedimenting at 52-55 S. It has not been characterized extensively, but attempts to demonstrate the presence of viral structural polypeptides therein have give uniformly negative results. The literature contains many reports of the isolation (between 24 and 48 hr postinfection) of viral DNA-protein complexes having sedimentation coefficients of the order of 50-60 S from cells infected with either SV40 or Py virus (9-U). Whether there is any relationship (structural or functional) between these complexes and the one described here remains to be seen. ACKNOWLEDGMENTS These studies were supported by grants to one of us (J. S. C.) from the Medical Research Council of Canada (MT 1191)and the National Cancer Institute of Canada. REFERENCES
1. COLTER,J. S., CHLUMECKA,V., and RALPH, R. K., Virology 70, 136-143 (1976). 2. MCCUTCHAN, J. H., and PAGANO,J. S.,J. Nat. Cancer Inst. 41, 351-357 (1968).
3. HIRT, B., J. Mol. Bid. 26, 365-369 (1967).
SHORT
COMMUNICATIONS
4. PENMAN, S. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.). Academic Press, New York, 1969. 5. FROST, E., and BOURGAUX, P., Virology 68,245255 (1975). 6. MACKAY, R. L., and CONSIGLI, R. A., J. Viral. 19, 620-636 (1976). 7. BARBANTI-BRODANO, G., SWETLY, P., and KoPROWSKI, H., J. Viral. 6, 78-86 (1970). 8. HUMMELER, K., TOMASSINI, N., and SOKOL, F., J. Viral. 6, 87-93 (1970). 9. GREEN, M. H., MILLER, H. I., and HENDLER, S.,
10. 11. 12. 13. 14.
223
Proc. Nat. Acad. Sci. USA 68, 1032-1036 (1971). WHITE, M., and EASON, R., J. Viral. 8, 363-371 (1971). SEEBECK, T., and WEIL, R., J. Viral. 13,567-576 (1974). SHMOOKLER, R. J., Buss, J., and GREEN, M. H., Virology 57, 122-127 (1974). MCMILLAN, J., and CONSIGLI, R. A. J. Viral. 14, 1326-1336 (1974). SHANI, M., SEIDMAN, M., and SALZMAN, N., Virology 83, 110-119 (1977).
94, 219-223
(197%
Early Events in the Infection of Permissive Comparison of Chymotrypsin-Treated
VERA CHLUMECKA, Department
of Biochemistry,
Cells with Polyoma Virus: and Untreated Virus
PERRY D’OBRENAN, University
of Alberta,
AND
Edmonton,
JOHN S. COLTERl
Alberta
T6G 2H7, Canada
Accepted December 5, 1978 An examination of the early events in the infection of cultured mouse embryo fibroblasts using both chymotrypsin-treated (chymo+) and untreated (chymo-) polyoma virus has shown that exposure to the protease has no effect on the ability of the virions to attach to, penetrate into, and enter the nuclei of cells. Uncoating, which our data indicate takes place exclusively in the nuclei, appears to be somewhat delayed in the case of the chymo+ virions. The only identifiable product of uncoating is a DNA protein complex having a sedimentation coefficient of the order of 52-55 S. Stimulation of cellular and viral DNA synthesis was found to be greatly reduced in cells infected with chymo+ virions relative to those infected with chvmo- virions. but the specific infectivities of DNAs isolated from chymo+ and chymovirions were found to be pre&ly the same.
It was reported earlier (2) that incubation of polyoma (Py) virus with chymotrypsin during purification results in a marked decrease in the recovery of infectious virus (PFU) but not of hemagglutinating activity (HAU), and that exposure to the protease has no effect on the physical integrity of either the virion or its DNA but leads to the appearance in the virions of a new polypeptide, apparently produced from VP3 by chymotryptic cleavage. We report here the results of an investigation in which Py virus exposed to chymotrypsin during purification (chymo+ virions) and Py virus purified without exposure to the protease (chymo- virions) were examined with respect to the early events of the infectious cycle. In addition, the abilities of chymo’ and chymo- virions to stimulate the synthesis of cellular and viral DNA were compared, as were the infectivities of DNAs isolated from the two preparations. Tertiary cultures of mouse embryo (ME) nbroblasts, used in all experiments described herein, were established and grown as described previously (1). Virus pools were prepared by infecting confluent mouse kidney cultures at a m.o.i. of 1 and harvesting when cell lysis was complete. To prepare labeled ’ To whom reprint
requests
should be sent. 219
virus, the medium on the infected cultures was suppIemented with [3H]thymidine (20 FCi/ml). The virus was purified as described previously (1). Virus pools were carried through the first three steps of the purification procedure (methanol precipitation, sequential treatment with receptor destroying enzyme (RDE), and RNase + DNase) and then divided into two equal aliquots, only one of which was incubated with a-chymotrypsin before the purification was completed. This led in each case to the production of two preparations (chymo+ and chymo-) that contained the same concentration of HAUs and physical particles, and, when the vii-ions were labeled, the same specific activity (counts per minute per physical particle). DNAs, isolated by phenol extraction from equal aliquots of chymo+ and chymo- virions, had infectivities [measured in ME monolayers using the DEAE-dextran procedure @)] of 1.4 x lo6 and 1.2 x lo6 PFU/ ml, respectively. Both contained 5 pg DNA/ ml. The corresponding virus suspensions had titers of 4.2 x 10s and 4.9 x lOlo PFU/ml. The synthesis of cellular and viral DNAs in cells infected with chymo+ and chymo0042~6822/79/050219-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
220
SHORT COMMUNICATIONS
FIG. 1. (Upper) Kinetics of attachment of labeled chymo- and chymo+ Py virions to ME cells at 22”. At each time, the amount of radioactivity that had become firmly cell associated was measured and expressed as a percentage of the input radioactivity. (0) chymovirions; (0) chymo+ virions. (Lower) Kinetics of penetration of labeled chymo- and chymo+ Py virions into ME cells. Cultures to which virus had been attached at 2” and then incubated at 37” were treated with RDE at various times postattachment. The amount of radioactivity not removed by RDE was expressed as a percentage of the radioactivity that was cell associated at zero time. (0) chymo- virions; (0) chymo+ virions.
virions at equal input multiplicities of physical particles (50 PFU/cell of chymo- virions) was examined by pulse labeling infected monolayer-s for 30 min with [14C]thymidine (2 &i/ml) at various times from 0 to 36 hr postattachment, fractionating the pulse-labeled monolayers by the Hirt procedure (3), and monitoring cellular and viral DNA synthesis by measuring the acid-insoluble radioactivity in the Hirt pellet and supernatant, respectively. It was found (data not shown) that the onset of synthesis was delayed (by lo-12 hr) and the extent of synthesis of both species was much depressed in cells infected with chymo+ virions.
To study attachment, replicate monolayers of ME cells were infected with labeled ehymo+ and chymo- virions at equal input multiplicities of physical particles (50 PFUl cell of chymo- virions) and incubated at 22”. The amount of radioactivity that had become firmly cell associated (not removed by washing with PBS) was determined at various times and expressed as a percentage of the input radioactivity (5 x lo4 cpmlplate in both series). To examine penetration, monolayers were incubated with labeled virions for 2 hr at 2”, after which they were washed and incubated in DME -2% fetal calf serum (FCS) at 37”. Periodically thereafter, cultures were treated with RDE (‘750units in 3 ml phosphate-buffered saline (PBS), 15min at 37”) to remove virus still attached to the plasma membrane. The extent of penetration was estimated from measurements of the fraction of the radioactivity that remained cell associated after RDE treatment. The results of these studies are illustrated in Fig. 1. The kinetics of attachment of chymo+ and chymo- virions to cells were found to be essentially identical (attachment reaches a plateau in 2-3 hr with 60-70% of the virions firmly cell associated) as were the rates at which chymo+ and chymo- virions, attached to cells at 2”, penetrate into the cells during a subsequent incubation at 37”. The initial rate of penetration was found to be quite rapid (about 50% within 1 hr), after which virus continues to enter the cells at a decreased rate until, by 6-8 hr, 85-90% of the cell-associated virions can no longer be removed by RDE. The intracellular distribution of virus as a function of time post-attachment was studied as follows. Monolayers were infected by incubation with labeled virus for 2 hr at 2”, after which they were washed with PBS and incubated at 37” in DME-2% FCS. At intervals thereafter, duplicate cultures were washed with PBS and incubated with RDE (see above), after which they were washed twice with NTM buffer (0.14 M NaCl, 1.5 mk’ MgCl,, 10 mM Tris, pH 7.4) and lysed by incubation for 10 min at 0” in the samebuffer containing 0.5% NP-40. After removal of the cytoplasmic fraction, the nuclei were
221
SHORT COMMUNICATIONS
washed with and collected into NTM buffer and disrupted by sonication. The amount of acid-insoluble radioactivity in each fraction was measured before and after incubation with DNase and expressed as a percentage of the total cell associated radioactivity at time zero (i.e., after attachment at 2’). Illustrative data are presented in Fig. 2. Several things are worth noting. The patterns of intracellular distribution obtained with the chymo+ and chymo- preparations are strikingly similar, with the rates of nuclear entry of the two being essentially identical. We find no evidence of any uncoating in the cytoplasm, using susceptibility of the viral genome to DNase as the criteria, and, during the first 6-8 hr postattachment, less of the nuclear radioactivity appears to be DNase-sensitive in chymo+ virion-infected cells than in chymo- virioninfected cells.
0
2
4 6 8 IO 12 INCUBATION TIME (hrs)
To obtain information regarding the fate of those v&ions that enter the nuclei, cells were infected with labeled virus, incubated, treated with RDE, and separated into cytoplasmic and nuclear fractions as outlined above. The nuclei were then treated briefly (5-10 see) with a mixture of sodium deoxycholate and Triton X-100 to remove outer nuclear membranes and cytoplasmic contamination (4) before being solubilized in 0.5 A4 NaCl, 0.05 M MgC&, 0.01 M Tris, pH 7.4. The preparations were homogenized briefly by hand (five strokes, Dounce homogenizer) before being analyzed by velocity sedimentation in linear lo-40% sucrose density gradients. The radioactivity was found to be resolved into (1) a rapidly sedimenting peak, (2) intact virions, and (3) a peak of more slowly sedimenting material with a sedimentation coefficient of the order of 52-55 S (see Fig. 2). When solubilized
14
16
IO 20 FRACTION
30 40 NUMBER
FIG. 2. (Left) Intracellular distribution of labeled chymo- (panel A) and chymo+ (panel B) Py vii-ions in ME cells. At various times postattachment, cultures were treated with RDE and separated into cytoplasmic and nuclear fractions. The amount of acid-insoluble radioactivity in each was measured before and after DNase treatment, and expressed as a percentage of the total cell-associated radioactivity at time zero. Circles, cytoplasmic counts per minute; squares, nuclear counts per minute; closed symbols, before DNase; open symbols, after DNase. (Right) Centrifugal analysis of solubilized nuclei isolated at 14 hr postattachment from ME cells infected with labeled chymo- (panel A) and chymo+ (panel B) Py virions. Centrifugation was for 90 min at 35,000rpm (SW41 rotor, Beckman L5-65 centrifuge) through lo-40% linear sucrose gradients. Sedimentation was from right to left. The arrows indicate the position of markers, Py virions (l), Mengo virions (2), R17 virions (3), and Py DNA I (4) in parallel gradients.
SHORT COMMUNICATIONS
222
TABLE 1
DISTRIBUTIONOFRADIOACTIVITYINSOLUBILIZEDNUCLEI ISOLATEDFROM CELLSINFECTED WITH[~H]THYMIDINE-LABELED CHYMO+ANDCHYMO-POLYOMAVIRION~ Chymo- Pyvirus Hours postattachment
Fastsedimenting component
Virion peak
2 6 10 14 24
41.2 41.5 35.0 33.6 39.0
52.1 41.3 35.0 33.9 26.4
Chymo+ Py virus
material
Fastsedimenting component
Virion peak
material
6.7 17.2 30.0 32.5 34.6
54.6 48.7 39.8 39.6 34.4
45.4 42.5 47.4 38.6 34.0
0.0 8.8 12.8 21.8 31.6
52-55 S
52-55 S
a Nuclei were isolated and solubilized, and the distribution of radioactivity therein was determined by centrifugal analysis as outlined in the legend to Fig. 2. The amount of radioactivity in each peak is expressed as a percentage of the total radioactivity recovered in the three peaks.
nuclei were incubated with DNase prior to centrifugation, a small amount of radioactivity was lost from the fast-sedimenting peak, the virion peak was unaffected, and the 5255 S peak was eliminated completely. The results of sedimentation analyses of solubilized nuclei isolated from chymo+ and chymo- virion-infected cells at various times from 2 to 24 hr postattachment are summarized in Table 1. The 52-55 S material can be detected in cells infected with chymovirions as early as 2 hr postattachment and increases progressively in amount during the 24-hr period. The appearance of this material was found to be delayed by 4-6 hr in cells infected with chymo+ virions, although by 24 hr postattachment it reaches levels comparable to those found in cells infected with chymo- virions. The data presented here show that exposure of Py virus to chymotrypsin during purification has no significant effect on the ability of the virions to attach to, penetrate into, and enter the nuclei of permissive cells. Exposure to the protease seems to decrease the efficiency with which the treated virions are uncoated, but this difference in the behavior of chymo+ and chymo- virions is transitory and does not provide a satisfactory explanation for the approximately 2 log loss in infectivity resulting from exposure to chymotrypsin. Our conclusion that the
uncoating of Py virus takes place exclusively in the nuclei is at variance with that of Frost and Bourgaux (5) but in agreement with that of Mackay and Consigli (6) and of other investigators (7, 8) who have examined the uncoating of SV40. The only product of uncoating that we have found is the material sedimenting at 52-55 S. It has not been characterized extensively, but attempts to demonstrate the presence of viral structural polypeptides therein have give uniformly negative results. The literature contains many reports of the isolation (between 24 and 48 hr postinfection) of viral DNA-protein complexes having sedimentation coefficients of the order of 50-60 S from cells infected with either SV40 or Py virus (9-U). Whether there is any relationship (structural or functional) between these complexes and the one described here remains to be seen. ACKNOWLEDGMENTS These studies were supported by grants to one of us (J. S. C.) from the Medical Research Council of Canada (MT 1191)and the National Cancer Institute of Canada. REFERENCES
1. COLTER,J. S., CHLUMECKA,V., and RALPH, R. K., Virology 70, 136-143 (1976). 2. MCCUTCHAN, J. H., and PAGANO,J. S.,J. Nat. Cancer Inst. 41, 351-357 (1968).
3. HIRT, B., J. Mol. Bid. 26, 365-369 (1967).
SHORT
COMMUNICATIONS
4. PENMAN, S. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.). Academic Press, New York, 1969. 5. FROST, E., and BOURGAUX, P., Virology 68,245255 (1975). 6. MACKAY, R. L., and CONSIGLI, R. A., J. Viral. 19, 620-636 (1976). 7. BARBANTI-BRODANO, G., SWETLY, P., and KoPROWSKI, H., J. Viral. 6, 78-86 (1970). 8. HUMMELER, K., TOMASSINI, N., and SOKOL, F., J. Viral. 6, 87-93 (1970). 9. GREEN, M. H., MILLER, H. I., and HENDLER, S.,
10. 11. 12. 13. 14.
223
Proc. Nat. Acad. Sci. USA 68, 1032-1036 (1971). WHITE, M., and EASON, R., J. Viral. 8, 363-371 (1971). SEEBECK, T., and WEIL, R., J. Viral. 13,567-576 (1974). SHMOOKLER, R. J., Buss, J., and GREEN, M. H., Virology 57, 122-127 (1974). MCMILLAN, J., and CONSIGLI, R. A. J. Viral. 14, 1326-1336 (1974). SHANI, M., SEIDMAN, M., and SALZMAN, N., Virology 83, 110-119 (1977).
