VIROLOGY
86, 281-286
Intracellular
(1978)
Forms of the Parental Human Cytomegalovirus Early Stages of the Infective Process
JONG-HO
The
Wistar
Institute
JEAN,
NORIAKI YOSHIMURA, TORU STANLEY A. PLOTKIN’
of Anatomy
and Biology and Children’s Pennsylvania 19104 Accepted
December
Hospital
Genome at
FURUKAWA, of Philadelphia,
AND
Philadelphia,
8, 1977
Intracellular forms of human cytomegalovirus (HCMV) DNA molecules isolated from infected cells were examined by electron microscopy before and after the onset of viral DNA synthesis. In cells harvested before showing replicative forms of viral DNA, circular and concatemeric molecules were observed in addition to linear double-stranded molecules, The observation of circular, unbranched, unit-size molecules suggests that HCMV DNA has a repetitive sequence that is located at or near the termini and is exposed by exonuclease digestion within infected cells, and that single-stranded regions can complement each other to form circles. Linear molecules (unit size or smaller) and concatemers with replicative “eye” loops or forks were observed after DNA replication could be assumed to have begun. Viral DNA molecules with terminal loops were also observed. These structures indicate that an inverted repetition of the sequence may be present within the terminal region. The functions of these molecules and of circular molecules are unknown.
Human cytomegalovirus (HCMV) has been suspected as an oncogenic virus due to its ability to stimulate host cell DNA synthesis in both permissive and abortive infections (I, 2) and due to its transforming capacity (3). Early events in the HCMV infective process are particularly interesting because decisions about the nature of a viral infection, i.e., whether it will be lytic, abortive, or transforming, are probably made at the preliminary stage. HCMV has an eclipse period of 48-72 hr (1,4, 5). The reason for this long latent period is unknown. No previous studies have described intracellular forms of HCMV DNA during this stage. Therefore, the present study was undertaken to characterize intracellular forms of parental HCMV DNA during the eclipse period before the onset of DNA synthesis in a lytic infection. In order to be certain that the experiments preparing parental DNA for electron
microscope (EM) observation would be performed before the onset of DNA synthesis, it was first necessary to perform an experiment to determine when viral DNA synthesis begins. For this preliminary experiment, confluent monolayer cultures of WI-38 cells were infected with HCMV at a multiplicity of infection (m.0.i.) of 0.1 plaque-forming units (PFU)/cell. One hour after infection, the cultures were refed with minimum essential medium (MEM) containing 2% fetal bovine serum. Twenty-four hours after infection, r3H]thymidine (5 &i/ml) was added to the medium of the culture. The cells were harvested, lysed with Sarkosyl, and digested with Pronase. The DNA aliquot was mixed with [‘4C]thymidine-labeled cellular DNA and centrifuged in CsCl gradients at 32,000 rpm for 64 hr to reach equilibrium. The CsCl gradients were fractionated and the radioactivity of each fraction was determined. Figure 1B shows that the [3H]thymidine taken up by cells from 24 to 45 hr after infection was almost exclusively incorporated into cellular DNA (density = 1.70 g/ml). NO [3H]thymidine incor-
‘To whom requests for reprints should be addressed at The Wistar Institute Anatomy and Biology, Philadelphia, Pennsylvania 19104. 281
0042-6822/78/0861-0281$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved
SHORT COMMUNICATIONS
3.0
to Table 1. To examine parental viral DNA by EM, 3H-labeled viral DNA was isolated before the frst round of viral DNA synthesis from cells that had been infected with [3H]thymidine-labeled virus in the second step of the experiment. On all operations, shear degradation of high molecular weight DNA was avoided, and only the viral DNA that had good separation from cellular DNA in the CsCl gradients was used for EM examination. Viral DNA was spread by the Kleinschmidt (6) technique which was slightly modified TABLE
2.0
1.5
Type of,D,cA
Unit
FRACTION
NUMBER
1. WI-38 cells were infected with HCMV. After adsorption the inoculum was removed and cells were incubated further in MEM. fienty-four hours after infection, cells were labeled with [3H]thymidine in the same medium. At 45 hr p.i. the cells were harvested, lysed with Sarkosyl, and digested with Pronase. (A) DNA from mature virions and “C-labeled cellular DNA. (B) Cellular DNA ([14C]thymidine) was added to the lysate and both were ultracentrifuged in CsCl. FIG.
porated into viral DNA could be detected up to this time. As shown in Fig. lA, C3H] thymidine-labeled viral DNA extracted separately from matured virions has a density of 1.72 g/ml and good separation from cellular DNA. The results confirm a previous observation, under the same conditions, that viral DNA synthesis does not begin until 48 hr postinfection (p.i.) (4). Since virus is labeled during replication, in order to obtain [3H]thymidine-labeled parental viral DNA for observation, a twostep experiment was necessary. The first step produced 3H-labeled virus that was then harvested from cells and used as an inoculum in the second step. Both steps of the experiment are described in the legend
1
FREQUENCY INTRACELLULAR
size,
DISTRIBUTION OF VARIOUS FORMS OF PARENTAL HCMV DNA MOLECULES~ mole- Number (or percentage) of Le;@F molecules* m
linear
molecules Unit size, circular molecules Molecules larger than unit length (concatemers) Molecules with terminal loop(s)
4 hr pi.
42 hr p.i.
366 (91%)
143 (80.8%)
4 (1%)
8 (4.5%)
48-52 46-52
13 (3%)
14 (8%)
68-92
20 (5%)
12 (6.7%)
50-77
(1[3H]Thymidine-labeled parental viral DNA was obtained for EM observation as follows: First, C3H]thymidine-labeled virus was prepared by infecting WI38 cells with HCMV (10 PFU/cell); 24 hr p.i. [3H]thymidine (10 &i/ml) was added and cells were further incubated. The virus was harvested at 7 days p.i. and purified (8). The titer of the ‘H-labeled virus, which was approximately 3-5 x lo6 (PFU/cell), was diluted one-tenth when used to infect WI-38 cells (3-5 x 106). 3H-Labeled parental viral DNA was prepared by infecting WI-38 cells with [3H]thymidine-labeled virions (0.1 PFU/cell) in MEM containing thymidine (100 gg/ml) and deoxycytidine (10 pg/ml). After adsorption the cells were washed eptensively to remove unadsorbed virus and were incubated further in the same medium. At 4 or 42 hr p.i., the cells were harvested, lysed with Sarkosyl (l%), heated at 45” for 15 min, and digested with Pronase (2.5 mg/ml). The lysate (equivalent to 106 cells) was centrifuged to equilibrium in CsCl in a Spinco SW 50.1 rotor at 32,600 rpm for 64 hr to separate 3H-labeled viral DNA from cellular DNA. b Only molecules of unit length or longer were included. Such molecules comprised approximately 65% of the DNA in the preparation.
SHORT
COMMUNICATIONS
by us as follows. The spreading solution we used contained 0.5 c(g of DNA, 0.05 mg/ml of cytochrome c, 0.1 M Tris-HCI (pH 8.4), 0.01 M Naz-EDTA, and 40% formamide. Forty microliters of this solution was spread on hypophase consisting of the same salts as the spreading solution and formamide of a lower concentration. Specimens were photographed by a Zeiss EM10 electron microscope. The electron micrographs were projected on paper, traced, and measured with a map measurer. Molecular weight of DNA was calculated by using SV40 DNA [molecular weight: 3.6 X lo6 (7)] as an internal standard. Up to 1.5 hr p.i., only linear, doublestranded DNA molecules of unit size were seen (Fig. 2). The DNA in mature virions treated similarly consisted of linear, doublestranded molecules. The contour length of the DNA (in 20 molecules from mature virions) measured in the experiment is predominantly in the range of 50 + 2 pm which suggests a molecular weight of 100 + 4 X 106. This is in good agreement with the weight estimated by velocity sedimentation in neutral gradients (8). Under the conditions used, by 42 hr p.i. no replicative forms of viral DNA could be detected. When intracellular viral DNA was isolated from infected cells at 4 and 42 hr p.i., three types of molecules were observed in addition to the linear, unit-size, double-stranded molecules. One was a closed, double-stranded, unit-size length molecule (Fig. 3) in the form of a relaxed circle, similar to molecules reported for parental pseudorabies virus DNA (9). A second type of molelcule was linear and longer than unit size. Single-stranded DNA does not spread well under the conditions used; however, linear molecules (unit size or longer than unit size) with a single-stranded region at either one or both ends were occasionally observed. These results suggest that linear, double-stranded parental viral DNA is digested by an exonuclease within the infected cells and that HCMV DNA exposes a repetitive sequence located at or near its termini, which can anneal intramolecularly to form circles. Concatemers have been observed during T4, T7, or pseudorabies viral replication (g-11). How-
283
ever, under the conditions we used, intermolecular alignment to form larger molecules before DNA replication is not likely. A recent report (12) describes a class of large viral DNA molecules, 150-155 x lo6 daltons, that form a small percentage of the total encapsidated viral DNA population. The concatemeric molecules of viral DNA observed before DNA replication in this study may be identified with the large molecules in this report. The third type of molecule was linear and double stranded with a terminal loop at one or both ends. The size of the terminal loop ranged from 0.2 to 0.5 pm. These structures indicate that, within the terminal region of the HCMV DNA, molecules may have an inverted repetition of sequences similar to those seen in HSV and pseudorabies virus DNA of the herpes virus group (5, 13).
The distribution of the different types of molecules obtained from the infected cells at 4 and 42 hr p.i. is tabulated in Table 1. As can be seen, by 42 hr there is an increase in the number (and percentage) of molecules forming circles or concatemers. The replication of a linear duplex molecule, logically, can only be completed through the formation of either circular or concatemeric replicative intermediates (14). Examination of the viral DNA molecules present within the infected cells after viral DNA synthesis began (53 hr p.i.) disclosed linear molecules (unit size or smaller) and concatemeric molecules with replicative “eye” loops or forks (4.5% of the 70 molecules examined). These results suggest that viral DNA molecules acquire single-stranded sequences and consequently form circles. The mechanisms for the formation of concatemers before DNA replication is not clear at present. Although the function of circles is still unknown, the important event to emerge from the finding of these unit-size, closed, circular molecules is the probable presence of reiterated sequences located at or near the termini of the viral DNA molecule. HCMV has been implicated as a possible oncogenic agent. It has been assumed that circularity of oncogenic virus DNA might be required for transformation (15). As mentioned in the beginning of the re-
284
SHORT
COMMUNICATIONS
FIG. 2. Electron micrograph of HCMV molecule. WI-38 cells were infected with [3H]thymidine-labeled virion and viral DNA was isolated as described in the legend to Table 1. Cells were harvested at 1.5 hr p.i. The DNA molecule measures 52 pm. The small cirr;ular molecule with the arrows is SV40 which serves as an internal marker for length measurement of DNA. Bar = 1 pm.
SHORT
COMMUNICATIONS
285
FIG. 3. Electron micrograph of circular HCMV DNA molecule. WI-38 cells were infected with I”H]thymidinelabeled virion and viral DNA was isolated as described in the legend of Table 1. Cells were harvested at 42 hr p.i. The DNA molecule measures 51.5 pm. Arrow indicates SV40 DNA molecule. Bar = 1 pm.
port, HCMV replication is extremely slow. Circular molecular formation in the viral infective process may be related to the virus’ potential for latent or persistent infection of the host.
The events in this study occur in the early stages of infection before and after the onset of viral DNA replication. Further studies to investigate the function and identity of these intracellular forms in abortive
286
SHORT
COMMUNICATIONS
or transformed cell cultures viously be significant.
would
ob-
ACKNOWLEDGMENTS This work was supported by USPHS Grant AI12151 from the National Institute of Allergy and Infectious Diseases and by RR-05540 from the Division of Research Resources. REFERENCES
1. ST. JEOR, S., ALBRECHT, T. B., FUNK, F. D., and RAPP, F., J. Viral. 13, 353-362 (1974). 2. FURUKAWA,
T., TANAKA,
S., and PLOTKIN,
S. A.,
Proc. Sot. Exp. Biol. Med. 148,211-214 (1975). 3. ALBRECHT, T., and RAPP, F., Virology 55, 53-61 (1973). 4. DEMARCHI,
J. M.,
and KAPLAN,
A.
S., J. Viral.
18,1063-1070(1976). 5. JEAN, J. H., BLANKENSHIP, M. L., and TAMAR, Virology 79, 281-291 (1977).
B.,
6. KLEINSCHMIDT, A. K., Methods Enzynol. 12B, 361-377 (1968). 7. TAI, H. T., SMITH, C. A., SHARP, P. A., and VINOGRAD, J., J. Virol. 9.317-325 (1972). 8. HUANG, E. S., CHEN, S. T., and PAGANO, J. S., J. Viral. 12, 1473-1481 (1973). 9. JEAN, J. H., ~~~TAMAR, B., Proc. Nut. Acad. Sci. USA 73,2674-2678 (1976). 10. MILLER, R. C., JR., Annu. Rev. Microbial. 29, 355-376 (1975). 11. BEN-P• RAT, T., KAPLAN, A. S., STEHN, B., and RUBENSTEIN, A. S., Virology 69, 547-560 (1976). 12. KILPATRICK, B. A., and HUANG, E., J. Viral, 24, 261-276 (1977). 13. WADSWORTH, S., HAYWARD, G. S., and RQIZMAN, B., J. ViroZ. 17,503-512 (1976). 14. WASTON, J. D., Nature New Biol. 239, 197-201 (1972). 15. DULBECCO, R., Science 142,932-936 (1963).
86, 281-286
Intracellular
(1978)
Forms of the Parental Human Cytomegalovirus Early Stages of the Infective Process
JONG-HO
The
Wistar
Institute
JEAN,
NORIAKI YOSHIMURA, TORU STANLEY A. PLOTKIN’
of Anatomy
and Biology and Children’s Pennsylvania 19104 Accepted
December
Hospital
Genome at
FURUKAWA, of Philadelphia,
AND
Philadelphia,
8, 1977
Intracellular forms of human cytomegalovirus (HCMV) DNA molecules isolated from infected cells were examined by electron microscopy before and after the onset of viral DNA synthesis. In cells harvested before showing replicative forms of viral DNA, circular and concatemeric molecules were observed in addition to linear double-stranded molecules, The observation of circular, unbranched, unit-size molecules suggests that HCMV DNA has a repetitive sequence that is located at or near the termini and is exposed by exonuclease digestion within infected cells, and that single-stranded regions can complement each other to form circles. Linear molecules (unit size or smaller) and concatemers with replicative “eye” loops or forks were observed after DNA replication could be assumed to have begun. Viral DNA molecules with terminal loops were also observed. These structures indicate that an inverted repetition of the sequence may be present within the terminal region. The functions of these molecules and of circular molecules are unknown.
Human cytomegalovirus (HCMV) has been suspected as an oncogenic virus due to its ability to stimulate host cell DNA synthesis in both permissive and abortive infections (I, 2) and due to its transforming capacity (3). Early events in the HCMV infective process are particularly interesting because decisions about the nature of a viral infection, i.e., whether it will be lytic, abortive, or transforming, are probably made at the preliminary stage. HCMV has an eclipse period of 48-72 hr (1,4, 5). The reason for this long latent period is unknown. No previous studies have described intracellular forms of HCMV DNA during this stage. Therefore, the present study was undertaken to characterize intracellular forms of parental HCMV DNA during the eclipse period before the onset of DNA synthesis in a lytic infection. In order to be certain that the experiments preparing parental DNA for electron
microscope (EM) observation would be performed before the onset of DNA synthesis, it was first necessary to perform an experiment to determine when viral DNA synthesis begins. For this preliminary experiment, confluent monolayer cultures of WI-38 cells were infected with HCMV at a multiplicity of infection (m.0.i.) of 0.1 plaque-forming units (PFU)/cell. One hour after infection, the cultures were refed with minimum essential medium (MEM) containing 2% fetal bovine serum. Twenty-four hours after infection, r3H]thymidine (5 &i/ml) was added to the medium of the culture. The cells were harvested, lysed with Sarkosyl, and digested with Pronase. The DNA aliquot was mixed with [‘4C]thymidine-labeled cellular DNA and centrifuged in CsCl gradients at 32,000 rpm for 64 hr to reach equilibrium. The CsCl gradients were fractionated and the radioactivity of each fraction was determined. Figure 1B shows that the [3H]thymidine taken up by cells from 24 to 45 hr after infection was almost exclusively incorporated into cellular DNA (density = 1.70 g/ml). NO [3H]thymidine incor-
‘To whom requests for reprints should be addressed at The Wistar Institute Anatomy and Biology, Philadelphia, Pennsylvania 19104. 281
0042-6822/78/0861-0281$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved
SHORT COMMUNICATIONS
3.0
to Table 1. To examine parental viral DNA by EM, 3H-labeled viral DNA was isolated before the frst round of viral DNA synthesis from cells that had been infected with [3H]thymidine-labeled virus in the second step of the experiment. On all operations, shear degradation of high molecular weight DNA was avoided, and only the viral DNA that had good separation from cellular DNA in the CsCl gradients was used for EM examination. Viral DNA was spread by the Kleinschmidt (6) technique which was slightly modified TABLE
2.0
1.5
Type of,D,cA
Unit
FRACTION
NUMBER
1. WI-38 cells were infected with HCMV. After adsorption the inoculum was removed and cells were incubated further in MEM. fienty-four hours after infection, cells were labeled with [3H]thymidine in the same medium. At 45 hr p.i. the cells were harvested, lysed with Sarkosyl, and digested with Pronase. (A) DNA from mature virions and “C-labeled cellular DNA. (B) Cellular DNA ([14C]thymidine) was added to the lysate and both were ultracentrifuged in CsCl. FIG.
porated into viral DNA could be detected up to this time. As shown in Fig. lA, C3H] thymidine-labeled viral DNA extracted separately from matured virions has a density of 1.72 g/ml and good separation from cellular DNA. The results confirm a previous observation, under the same conditions, that viral DNA synthesis does not begin until 48 hr postinfection (p.i.) (4). Since virus is labeled during replication, in order to obtain [3H]thymidine-labeled parental viral DNA for observation, a twostep experiment was necessary. The first step produced 3H-labeled virus that was then harvested from cells and used as an inoculum in the second step. Both steps of the experiment are described in the legend
1
FREQUENCY INTRACELLULAR
size,
DISTRIBUTION OF VARIOUS FORMS OF PARENTAL HCMV DNA MOLECULES~ mole- Number (or percentage) of Le;@F molecules* m
linear
molecules Unit size, circular molecules Molecules larger than unit length (concatemers) Molecules with terminal loop(s)
4 hr pi.
42 hr p.i.
366 (91%)
143 (80.8%)
4 (1%)
8 (4.5%)
48-52 46-52
13 (3%)
14 (8%)
68-92
20 (5%)
12 (6.7%)
50-77
(1[3H]Thymidine-labeled parental viral DNA was obtained for EM observation as follows: First, C3H]thymidine-labeled virus was prepared by infecting WI38 cells with HCMV (10 PFU/cell); 24 hr p.i. [3H]thymidine (10 &i/ml) was added and cells were further incubated. The virus was harvested at 7 days p.i. and purified (8). The titer of the ‘H-labeled virus, which was approximately 3-5 x lo6 (PFU/cell), was diluted one-tenth when used to infect WI-38 cells (3-5 x 106). 3H-Labeled parental viral DNA was prepared by infecting WI-38 cells with [3H]thymidine-labeled virions (0.1 PFU/cell) in MEM containing thymidine (100 gg/ml) and deoxycytidine (10 pg/ml). After adsorption the cells were washed eptensively to remove unadsorbed virus and were incubated further in the same medium. At 4 or 42 hr p.i., the cells were harvested, lysed with Sarkosyl (l%), heated at 45” for 15 min, and digested with Pronase (2.5 mg/ml). The lysate (equivalent to 106 cells) was centrifuged to equilibrium in CsCl in a Spinco SW 50.1 rotor at 32,600 rpm for 64 hr to separate 3H-labeled viral DNA from cellular DNA. b Only molecules of unit length or longer were included. Such molecules comprised approximately 65% of the DNA in the preparation.
SHORT
COMMUNICATIONS
by us as follows. The spreading solution we used contained 0.5 c(g of DNA, 0.05 mg/ml of cytochrome c, 0.1 M Tris-HCI (pH 8.4), 0.01 M Naz-EDTA, and 40% formamide. Forty microliters of this solution was spread on hypophase consisting of the same salts as the spreading solution and formamide of a lower concentration. Specimens were photographed by a Zeiss EM10 electron microscope. The electron micrographs were projected on paper, traced, and measured with a map measurer. Molecular weight of DNA was calculated by using SV40 DNA [molecular weight: 3.6 X lo6 (7)] as an internal standard. Up to 1.5 hr p.i., only linear, doublestranded DNA molecules of unit size were seen (Fig. 2). The DNA in mature virions treated similarly consisted of linear, doublestranded molecules. The contour length of the DNA (in 20 molecules from mature virions) measured in the experiment is predominantly in the range of 50 + 2 pm which suggests a molecular weight of 100 + 4 X 106. This is in good agreement with the weight estimated by velocity sedimentation in neutral gradients (8). Under the conditions used, by 42 hr p.i. no replicative forms of viral DNA could be detected. When intracellular viral DNA was isolated from infected cells at 4 and 42 hr p.i., three types of molecules were observed in addition to the linear, unit-size, double-stranded molecules. One was a closed, double-stranded, unit-size length molecule (Fig. 3) in the form of a relaxed circle, similar to molecules reported for parental pseudorabies virus DNA (9). A second type of molelcule was linear and longer than unit size. Single-stranded DNA does not spread well under the conditions used; however, linear molecules (unit size or longer than unit size) with a single-stranded region at either one or both ends were occasionally observed. These results suggest that linear, double-stranded parental viral DNA is digested by an exonuclease within the infected cells and that HCMV DNA exposes a repetitive sequence located at or near its termini, which can anneal intramolecularly to form circles. Concatemers have been observed during T4, T7, or pseudorabies viral replication (g-11). How-
283
ever, under the conditions we used, intermolecular alignment to form larger molecules before DNA replication is not likely. A recent report (12) describes a class of large viral DNA molecules, 150-155 x lo6 daltons, that form a small percentage of the total encapsidated viral DNA population. The concatemeric molecules of viral DNA observed before DNA replication in this study may be identified with the large molecules in this report. The third type of molecule was linear and double stranded with a terminal loop at one or both ends. The size of the terminal loop ranged from 0.2 to 0.5 pm. These structures indicate that, within the terminal region of the HCMV DNA, molecules may have an inverted repetition of sequences similar to those seen in HSV and pseudorabies virus DNA of the herpes virus group (5, 13).
The distribution of the different types of molecules obtained from the infected cells at 4 and 42 hr p.i. is tabulated in Table 1. As can be seen, by 42 hr there is an increase in the number (and percentage) of molecules forming circles or concatemers. The replication of a linear duplex molecule, logically, can only be completed through the formation of either circular or concatemeric replicative intermediates (14). Examination of the viral DNA molecules present within the infected cells after viral DNA synthesis began (53 hr p.i.) disclosed linear molecules (unit size or smaller) and concatemeric molecules with replicative “eye” loops or forks (4.5% of the 70 molecules examined). These results suggest that viral DNA molecules acquire single-stranded sequences and consequently form circles. The mechanisms for the formation of concatemers before DNA replication is not clear at present. Although the function of circles is still unknown, the important event to emerge from the finding of these unit-size, closed, circular molecules is the probable presence of reiterated sequences located at or near the termini of the viral DNA molecule. HCMV has been implicated as a possible oncogenic agent. It has been assumed that circularity of oncogenic virus DNA might be required for transformation (15). As mentioned in the beginning of the re-
284
SHORT
COMMUNICATIONS
FIG. 2. Electron micrograph of HCMV molecule. WI-38 cells were infected with [3H]thymidine-labeled virion and viral DNA was isolated as described in the legend to Table 1. Cells were harvested at 1.5 hr p.i. The DNA molecule measures 52 pm. The small cirr;ular molecule with the arrows is SV40 which serves as an internal marker for length measurement of DNA. Bar = 1 pm.
SHORT
COMMUNICATIONS
285
FIG. 3. Electron micrograph of circular HCMV DNA molecule. WI-38 cells were infected with I”H]thymidinelabeled virion and viral DNA was isolated as described in the legend of Table 1. Cells were harvested at 42 hr p.i. The DNA molecule measures 51.5 pm. Arrow indicates SV40 DNA molecule. Bar = 1 pm.
port, HCMV replication is extremely slow. Circular molecular formation in the viral infective process may be related to the virus’ potential for latent or persistent infection of the host.
The events in this study occur in the early stages of infection before and after the onset of viral DNA replication. Further studies to investigate the function and identity of these intracellular forms in abortive
286
SHORT
COMMUNICATIONS
or transformed cell cultures viously be significant.
would
ob-
ACKNOWLEDGMENTS This work was supported by USPHS Grant AI12151 from the National Institute of Allergy and Infectious Diseases and by RR-05540 from the Division of Research Resources. REFERENCES
1. ST. JEOR, S., ALBRECHT, T. B., FUNK, F. D., and RAPP, F., J. Viral. 13, 353-362 (1974). 2. FURUKAWA,
T., TANAKA,
S., and PLOTKIN,
S. A.,
Proc. Sot. Exp. Biol. Med. 148,211-214 (1975). 3. ALBRECHT, T., and RAPP, F., Virology 55, 53-61 (1973). 4. DEMARCHI,
J. M.,
and KAPLAN,
A.
S., J. Viral.
18,1063-1070(1976). 5. JEAN, J. H., BLANKENSHIP, M. L., and TAMAR, Virology 79, 281-291 (1977).
B.,
6. KLEINSCHMIDT, A. K., Methods Enzynol. 12B, 361-377 (1968). 7. TAI, H. T., SMITH, C. A., SHARP, P. A., and VINOGRAD, J., J. Virol. 9.317-325 (1972). 8. HUANG, E. S., CHEN, S. T., and PAGANO, J. S., J. Viral. 12, 1473-1481 (1973). 9. JEAN, J. H., ~~~TAMAR, B., Proc. Nut. Acad. Sci. USA 73,2674-2678 (1976). 10. MILLER, R. C., JR., Annu. Rev. Microbial. 29, 355-376 (1975). 11. BEN-P• RAT, T., KAPLAN, A. S., STEHN, B., and RUBENSTEIN, A. S., Virology 69, 547-560 (1976). 12. KILPATRICK, B. A., and HUANG, E., J. Viral, 24, 261-276 (1977). 13. WADSWORTH, S., HAYWARD, G. S., and RQIZMAN, B., J. ViroZ. 17,503-512 (1976). 14. WASTON, J. D., Nature New Biol. 239, 197-201 (1972). 15. DULBECCO, R., Science 142,932-936 (1963).
