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Multiplication of polyoma virus in cells of a continuous hamster line susceptible to transformation
DISCUSSION
120
AND
PRELIMINARY
bodies suggeststhe possibility of sectioning through a helical structure. In summary, addition of FPA with PA to growth media of FL cells infected with HSV has resulted in marked accumulations of fibrils, diplosomes, and spherical bodies. Examination of FL cells from three control cultures (consisting of “A”, FL cells and FPA; “B” FL cells alone; and “D”, FL cells and HSV) failed to show any of these previously undescribed morphological forms. Probably the most important problem to be resolved is that of identification of the biochemical composition of the viral structures and subunits which are observed in the micrographs, since interpretations of the sequential events in replication processesare quite dependent upon this information. Work in progress concerns applications of selective enzymatic digests of serial sections of the cells, particularly with proteinases, RNase, and DNase. These sectionsare being compared by means of electron microscopy with each other and with undigested control sections, providing the means for inferential identification of structures. Further investigations are being directed toward ascertainment of whether the phenomena observed are sufficiently universal in effect, to include intracytoplasmic viruses (vaccinia) or RNA viruses in the picornavirus group. A. CHITWOOD E. C. BRaCKEX’ (with the technical R. S. Scott) 1,.
Department Children’s Department University Oklahoma Accepted
assistance of
of Pediatrics Memorial Hospital, and of Microbiology Oklahoma Medical Center City, Oklahoma June 9, 1964
of
2 Supported by a Career Development Fellowship Award from the National Institutes of Health.
Multiplication
of
Continuous
Polyoma
Hamster
Virus Line
in
Susceptible
Cells
of
a
to
Transformation
types of responseare shown by cells exposed to polyoma virus in tissue Two
different
REPORTS
culture: cytopathic effect resulting from virus multiplication (1, .2), and transformation to altered growth characteristics of a small proportion of the cell population 6%4). In BHK21 hamster fibroblasts (4), only the second type of reaction can be easily detected (3, 4) and the viability of the whole cell population, judged by several criteria, is unaltered by exposure to virus. Although it is clear that large amounts of virus are not produced in this system (3, 5) virus synthesis at a low level would not be detected by the usual methods because of residual virus inoculum. However, fluorescent antibody studies (6) have shown appearance of virus specific antigen, suggestive of virus synthesis, in the nuclei of a small proportion of BHK21 cells, 34 days after exposure to polyoma virus. This paper reports the results of experiments using isotope incorporation to detect synthesis of new virus particles. Half-confluent monolayers of BHK21 cells were washed with saline solution and incubated for 24 hours in phosphate free Eagle’s medium containing 5 % dialyzed calf serum. Twenty-four hours later, the medium was replaced with fresh medium containing P32 (150 PC/ml, carrier-free orthophosphate). After 48 hours’ further incubation, the cells, removed from the glasswith Versene (0.0005 M), were exposed in suspension to polyoma virus, as in the standard transformation assay (3) and plated again in phosphate-free medium (50 PCof P32per milliliter). Very little further growth was observed. Four days after exposure to virus, the cells were harvested and mixed with nonradioactive virus, in the form of secondary cultures of whole mouse embryos infected 5 days previously with polyoma virus to act as carrier and marker virus. Virus was extracted with receptor-destroying enzyme (7) from the pooled cells, purified by a procedure already described (8) using ribonuclease and deoxyribonuclease, and examined for the presence of radioactive
material
with
the physical
properties
of polyoma virus. Extraction
of the preparation
with
phenol
121
FIG. 1. The distribution of radioactivity and carrier virus hemagglutinating activity after cquilibrium density centrifugation {A), velocity centrifugation (B), and zone electrophoresis (C) of the radioactive preparation. (A) Centrifugation for 18 hours at 30,000 rpm (Spinco SW 39L rotor) in buffered rubidium chloride (density 1.3 g/ml). (R) Centrifugation on a 20/5% BUCIPOS~ gradient in pH 7.2,0.02 M phosphate buffer for 25 minutes at 35,ooO rpm (Spinco SW 391; Rotar). The gradients (A, B) were fraetionated by puncturing the bottom of the tube and collecting drops, {C) ~~ec~rophoresis in a column aontaining a sucrose gradient (204%) in pH 7.2, 0.02 M phosphate buffer for 5 hours at 5 volts/cm. One-miIliliter fractious were collected. The radioa&tivi~y (a-----e, ~ou~ts~rnin.~ and hen~agglutinat~ng activity, expressed as the reciprocal of the dilution causing partial hema~glutination
122
DISCUSSION
AND
PRELIMINARY
gave trichloroacetic acid-insoluble radioactive material, which became acid soluble after treatment with deoxyribonuclease (100 erg/ml in 0.05 M MgC&) suggesting the presence of radioactive material in the form of DNA. Equilibrium density gradient centrifugation (Fig. 1) gave a peak in the region of the “full” particles (9) of the carrier virus corresponding to f13 % of the total radioactivity, but a radioactive background spread throughout the gradient showed that other material was also present. Velocity centrifugation in a sucrose gradient (Fig. 1) demonstrated a peak of radioactivity, comprising about 10 % of the total, sedimenting in the position of the faster hemagglut~at~g ~oin~nent of polyoma virus. A significant proportion of the total radioactivity (14%) could be adsorbed to and eluted from guinea pig red blood cells (Table 1), under the same conditions as polyoma virus hema~lutinin (7). TABLE
1
ADSORPTION OF THE RADIOACTIVE TO GUINEA PIG RED BLOOD
Fraction
pH
PREPARATION CELLS~
‘T‘--rperZGU acore -A~ (“C)
Nonadsorbed radioactivity First wash Second wash Third wash Eluted radioactivity
Counts/ min./fraction (background)
6.5
4
602
6.5 6.5 6.5 8.5
4 4 4 37
20 5 0 103
a A sample of the radioactive preparation at pH 6.5 was mixed with guinea pig red blood cells (final concentration 10o/O). The mixture was agitated for 30 minutes at 4” and centrifuged; the red cell peilet was washed three times with cold saline solution containing phosphate buffer (pH 6.5, 0.02 M). The cells were finally suspended in phosphate buffer pH 8.5 (0.04 M in saline), shaken for 30 minutes at 37”, and sedimented. The radioactivity of the supernatants was measured.
REPORTS
Finally, a sample of the radioactive preparation, dialyzed against phosphate buffer (pH 7.2, 0.02 IM) overnight at 4°C was subjected to zone electrophoresis with the apparatus described by Thorne (10). About 70 % of the total r~ioacti~ity migrated at the speed of polyoma virus; the higher proportion of viruslike material present in this case may result from losses of contaminating radioactivity during dialysis (Fig. 1). In the absence of biological studies, the results presented suggest that BHKZl cells, or at least a fraction of the cell population, when exposed to high multiplicity of virus infection, are capable of producing material which has the physical properties of polyoma virus. It is not known whether the production of viral material is directly related to the viral antigen synthesis revealed by immunofluorescence (6) or to the phenomenon of transformation. REFERENCES 1. EDDY, B. E., STEWART, S. E., and BERHELEY, W., Proc. Sot. Exptl. Biol. Med. 98, 848851 (1958). 1. SACHS, L., FOGEL, M., and WINOCOUR, E., Naturs 183, 663-664 (1959). S. STOEER, M., and ABEL, P., Cold Spring Harbor SyTnp. Quant. BioE. 27, 376-386 (1962). 4. MACPWERSON, I., and STOB;ER, M., Virology 16, 147-151 (1962). 6. FRASER, IL B., and CRAWFORD, E. M., unpublished. 6. FRASER, K. B., and GKARPURE, M., Virology 18, 505-507 (1962). 7. CRAWFORD, L. V., ~~~~0~0~~ 18, 177-181 (1962). 8. BOURGAUX, P., Virology 23,46-55 (1964). 9. CRAWFORD, L. V., CRAWFORD, E. M., and WATSON, D. H., Virology 18, 170-176 (1962). IO. THORNE, 1%. V., J. Bacterial. 85, 1247-1255 (1963). P. BOJZR~.LUX’ institute of Virology University of Glasgow Glasgow, Scotland Accepted June 9, 1964 1 Present Universite
address: Laboratoire de Bacteriologic, de Bruxelles, Bruxelles, Belgium.
120
AND
PRELIMINARY
bodies suggeststhe possibility of sectioning through a helical structure. In summary, addition of FPA with PA to growth media of FL cells infected with HSV has resulted in marked accumulations of fibrils, diplosomes, and spherical bodies. Examination of FL cells from three control cultures (consisting of “A”, FL cells and FPA; “B” FL cells alone; and “D”, FL cells and HSV) failed to show any of these previously undescribed morphological forms. Probably the most important problem to be resolved is that of identification of the biochemical composition of the viral structures and subunits which are observed in the micrographs, since interpretations of the sequential events in replication processesare quite dependent upon this information. Work in progress concerns applications of selective enzymatic digests of serial sections of the cells, particularly with proteinases, RNase, and DNase. These sectionsare being compared by means of electron microscopy with each other and with undigested control sections, providing the means for inferential identification of structures. Further investigations are being directed toward ascertainment of whether the phenomena observed are sufficiently universal in effect, to include intracytoplasmic viruses (vaccinia) or RNA viruses in the picornavirus group. A. CHITWOOD E. C. BRaCKEX’ (with the technical R. S. Scott) 1,.
Department Children’s Department University Oklahoma Accepted
assistance of
of Pediatrics Memorial Hospital, and of Microbiology Oklahoma Medical Center City, Oklahoma June 9, 1964
of
2 Supported by a Career Development Fellowship Award from the National Institutes of Health.
Multiplication
of
Continuous
Polyoma
Hamster
Virus Line
in
Susceptible
Cells
of
a
to
Transformation
types of responseare shown by cells exposed to polyoma virus in tissue Two
different
REPORTS
culture: cytopathic effect resulting from virus multiplication (1, .2), and transformation to altered growth characteristics of a small proportion of the cell population 6%4). In BHK21 hamster fibroblasts (4), only the second type of reaction can be easily detected (3, 4) and the viability of the whole cell population, judged by several criteria, is unaltered by exposure to virus. Although it is clear that large amounts of virus are not produced in this system (3, 5) virus synthesis at a low level would not be detected by the usual methods because of residual virus inoculum. However, fluorescent antibody studies (6) have shown appearance of virus specific antigen, suggestive of virus synthesis, in the nuclei of a small proportion of BHK21 cells, 34 days after exposure to polyoma virus. This paper reports the results of experiments using isotope incorporation to detect synthesis of new virus particles. Half-confluent monolayers of BHK21 cells were washed with saline solution and incubated for 24 hours in phosphate free Eagle’s medium containing 5 % dialyzed calf serum. Twenty-four hours later, the medium was replaced with fresh medium containing P32 (150 PC/ml, carrier-free orthophosphate). After 48 hours’ further incubation, the cells, removed from the glasswith Versene (0.0005 M), were exposed in suspension to polyoma virus, as in the standard transformation assay (3) and plated again in phosphate-free medium (50 PCof P32per milliliter). Very little further growth was observed. Four days after exposure to virus, the cells were harvested and mixed with nonradioactive virus, in the form of secondary cultures of whole mouse embryos infected 5 days previously with polyoma virus to act as carrier and marker virus. Virus was extracted with receptor-destroying enzyme (7) from the pooled cells, purified by a procedure already described (8) using ribonuclease and deoxyribonuclease, and examined for the presence of radioactive
material
with
the physical
properties
of polyoma virus. Extraction
of the preparation
with
phenol
121
FIG. 1. The distribution of radioactivity and carrier virus hemagglutinating activity after cquilibrium density centrifugation {A), velocity centrifugation (B), and zone electrophoresis (C) of the radioactive preparation. (A) Centrifugation for 18 hours at 30,000 rpm (Spinco SW 39L rotor) in buffered rubidium chloride (density 1.3 g/ml). (R) Centrifugation on a 20/5% BUCIPOS~ gradient in pH 7.2,0.02 M phosphate buffer for 25 minutes at 35,ooO rpm (Spinco SW 391; Rotar). The gradients (A, B) were fraetionated by puncturing the bottom of the tube and collecting drops, {C) ~~ec~rophoresis in a column aontaining a sucrose gradient (204%) in pH 7.2, 0.02 M phosphate buffer for 5 hours at 5 volts/cm. One-miIliliter fractious were collected. The radioa&tivi~y (a-----e, ~ou~ts~rnin.~ and hen~agglutinat~ng activity, expressed as the reciprocal of the dilution causing partial hema~glutination
122
DISCUSSION
AND
PRELIMINARY
gave trichloroacetic acid-insoluble radioactive material, which became acid soluble after treatment with deoxyribonuclease (100 erg/ml in 0.05 M MgC&) suggesting the presence of radioactive material in the form of DNA. Equilibrium density gradient centrifugation (Fig. 1) gave a peak in the region of the “full” particles (9) of the carrier virus corresponding to f13 % of the total radioactivity, but a radioactive background spread throughout the gradient showed that other material was also present. Velocity centrifugation in a sucrose gradient (Fig. 1) demonstrated a peak of radioactivity, comprising about 10 % of the total, sedimenting in the position of the faster hemagglut~at~g ~oin~nent of polyoma virus. A significant proportion of the total radioactivity (14%) could be adsorbed to and eluted from guinea pig red blood cells (Table 1), under the same conditions as polyoma virus hema~lutinin (7). TABLE
1
ADSORPTION OF THE RADIOACTIVE TO GUINEA PIG RED BLOOD
Fraction
pH
PREPARATION CELLS~
‘T‘--rperZGU acore -A~ (“C)
Nonadsorbed radioactivity First wash Second wash Third wash Eluted radioactivity
Counts/ min./fraction (background)
6.5
4
602
6.5 6.5 6.5 8.5
4 4 4 37
20 5 0 103
a A sample of the radioactive preparation at pH 6.5 was mixed with guinea pig red blood cells (final concentration 10o/O). The mixture was agitated for 30 minutes at 4” and centrifuged; the red cell peilet was washed three times with cold saline solution containing phosphate buffer (pH 6.5, 0.02 M). The cells were finally suspended in phosphate buffer pH 8.5 (0.04 M in saline), shaken for 30 minutes at 37”, and sedimented. The radioactivity of the supernatants was measured.
REPORTS
Finally, a sample of the radioactive preparation, dialyzed against phosphate buffer (pH 7.2, 0.02 IM) overnight at 4°C was subjected to zone electrophoresis with the apparatus described by Thorne (10). About 70 % of the total r~ioacti~ity migrated at the speed of polyoma virus; the higher proportion of viruslike material present in this case may result from losses of contaminating radioactivity during dialysis (Fig. 1). In the absence of biological studies, the results presented suggest that BHKZl cells, or at least a fraction of the cell population, when exposed to high multiplicity of virus infection, are capable of producing material which has the physical properties of polyoma virus. It is not known whether the production of viral material is directly related to the viral antigen synthesis revealed by immunofluorescence (6) or to the phenomenon of transformation. REFERENCES 1. EDDY, B. E., STEWART, S. E., and BERHELEY, W., Proc. Sot. Exptl. Biol. Med. 98, 848851 (1958). 1. SACHS, L., FOGEL, M., and WINOCOUR, E., Naturs 183, 663-664 (1959). S. STOEER, M., and ABEL, P., Cold Spring Harbor SyTnp. Quant. BioE. 27, 376-386 (1962). 4. MACPWERSON, I., and STOB;ER, M., Virology 16, 147-151 (1962). 6. FRASER, IL B., and CRAWFORD, E. M., unpublished. 6. FRASER, K. B., and GKARPURE, M., Virology 18, 505-507 (1962). 7. CRAWFORD, L. V., ~~~~0~0~~ 18, 177-181 (1962). 8. BOURGAUX, P., Virology 23,46-55 (1964). 9. CRAWFORD, L. V., CRAWFORD, E. M., and WATSON, D. H., Virology 18, 170-176 (1962). IO. THORNE, 1%. V., J. Bacterial. 85, 1247-1255 (1963). P. BOJZR~.LUX’ institute of Virology University of Glasgow Glasgow, Scotland Accepted June 9, 1964 1 Present Universite
address: Laboratoire de Bacteriologic, de Bruxelles, Bruxelles, Belgium.