VIROLOGY
71, 609-614 (1976)
Chromosomal DNA Replication: Retarded Fork Progression and Altered Initiation in Cells Infected with Wlengovirus or Newcastle Disease Virus ROGER HAND Departments
of Medicine
and Microbiology,
McGill
University,
Accepted January
Montreal,
Quebec H3A 2B4, Canada
13,1976
Infection of mouse L cells with mengovirus or Newcastle disease virus inhibits DNA synthesis. The effects of this inhibition on chromosome replication have now been investigated using DNA fiber autoradiography, a technique permitting analysis of events on clusters of active replication units. At 5 hr after infection at high multiplicity with either virus, DNA replication is normal despite a marked inhibition of [3H]thymidine incorporation into DNA. By 6 hr, virus infection slows the rate of replication fork progression. In addition, virus infection alters the pattern of initiation of DNA replication in that the proportion of units with bidirectional replication is reduced and the usual synchrony of initiation events on clusters of units is decreased.
Infection of mouse L-929 cells productively with mengovirus, a picornavirus, or abortively with Newcastle disease virus (NDV), a paramyxovirus, results in the inhibition of incorporation of 13Hlthymidine into cellular DNA (l-3). Since neither virus alters the percentage of cells in the DNA synthetic phase of the cell cycle (4) or significantly slows the rate of replication fork progression (daughter chain elongation) (5, 61, both viruses are thought to block the multifocal initiation of DNA synthesis. The normal patterns of DNA initiation in mammalian cells have been described. Initiation occurs synchronously on clusters of replication units along the chromosome f7,8). Replication proceeds bidirectionally from the origins of these units, and the newly replicated chains eventually fuse with daughter chains from adjacent units (g-11). Inhibition of cellular protein synthesis with the antibiotics puromycin and cycloheximide alters these patterns (12). The frequency of initiation and the synchrony of initiation events on clusters is decreased and the proportion of units with bidirectional replication is reduced. In addition, these antibiotics inhibit repli-
cation fork progression (6, 11, 13-15). Since the effects of both mengovirus and NDV on DNA replication have been attributed to the concomitant inhibition of cellular protein synthesis (5, 6), it would be of interest to know whether these viruses alter initiation patterns in the same fashion as the antibiotics and under what circumstances, if any, they inhibit replication fork progression. In the present experiments, DNA fiber autoradiography, a technique that permits analysis of events on active replication units on the chromosome, has been used to analyze DNA replication patterns in infected cells. At 5 hr after infection, rate of replication fork progression, the incidence of bidirectional replication, and the synchrony of initiation in infected cells and controls are similar. By 6 hr after infection, all three parameters are decreased in infected cells. Monolayers of L-929 cells were infected with mengovirus, small plaque variant (16) at a m.o.i. of 200 PFU/cell or NDV, Hickman strain (2) at a m.o.i. of 500 PFU/ cell. The characteristics and the conditions for propagation of the viruses and cell lines have been described (5, 12). Adsorption was for 1 hr in one-tenth the usual amount 609
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of medium. Control cells were sham-infected. At 5 and 6 hr after infection, thymidine incorporation into DNA was determined in control and infected cells by pulse-labeling them with [3H]thymidine (Table 1, column 2). Thymidine incorporation into DNA is inhibited in infected cells. Under these experimental conditions, mengovirus-infected cells have normal morphology 5 hr after infection. By 6 hr after infection, cell rounding is present in lo-20% of the cells. NDV-infected cells show normal morphology through 7 hr after infection. [3H]leucine incorporation into proteins is decreased 6 hr after infection with either virus. Incorporation is 41.9% of controls in mengovirus-infected cells and 37.1% of controls in NDV-infected cells. The degree of inhibition of incorporation of thymidine and leucine into DNA and protein is comparable to that reported by others (1, 5, 17,18). Replicate monolayers of control and infected cells were pulse-labeled with TABLE DNA Experiment
1. 5-hr postinfection Control Mengo-infected NDV-infected 2. 6-hr postinfection Control Mengo-infected NDV-infected
13Hlthymidine and their DNA processed for fiber autoradiography (7,ll). This involved treatment of the cells with fluorodeoxyuridine (2 x 10e6M) for 30 min before the pulse to exhaust their endogenous thymidine nucleotide pools, and sequential labeling with 13Hlthymidine at high specific activity (50 Ci/mmole, 5 X lo+ M, the hot pulse) and low specific activity (5 Ci/mmole, 5.5 x lo-” M, the warm pulse) for 30 min each. Fluorodeoxyuridine remained in the medium during the pulses. The times postinfection indicated in Table 1, column 1, represent the midpoints of the hot pulse. At the completion of the pulses, the DNA was processed for fiber autoradiography by lysing the cells gently and spreading the DNA fibers on a glass slide. The preparations were dried and fixed, and then coated with light-sensitive emulsion. After 3-6 months exposure, the slides were developed and examined by light microscopy. Figure 1 shows several autoradiograms 1
REPLICATION IN VIRUS-INFECTED CELLS
PHlthymidine incor orationa (% 0 P control)
Rate of fork progression* (pm/min t SEM)
Bidirectional replication units, proportionc
Initiation synchrony (pairs of units with like patterns), proportiond
100.0 37.6 46.0
0.72 + 0.043 0.67 2 0.034 0.69 k 0.036
0.82 0.75 0.76
0.78 0.76 0.78
100.0 5.6 21.4
0.64 + 0.023 0.24 2 0.015 0.35 k 0.017’
0.78 0.65’ 0.64’
0.77 0.53’ 0.59
n Measured in replicate monolayers handled in an identical manner to control and experimental cells except that in lieu of undergoing the DNA fiber autoradiography protocol, the cells were pulse-labeled with [“Hlthymidine (0.5 &i/ml) for 10 min at 5 or 6 hr after infection. Radioactivity incorporated into DNA was determined by acid precipitation and liquid scintillation counting. b Determined by measuring hot pulse track lengths of prepulse units in DNA sequentially labeled during a 30-min hot and 30-min warm pulse. On such tracks, the beginning and end of the hot pulse are marked. The length is divided by the duration of the hot pulse to obtain micrometers per minute. Fifty tracks were measured for each datum. c Determined by scoring internal replication units as to whether they showed unidirectional or bidirectional replication. DNA was labeled during a 30-min hot and 30-min warm pulse. One hundred units were scored for each datum. d Determined by scoring two-unit autoradiograms as to whether they contained a like pair or unlike pair of replication units. DNA was labeled during a 30-min hot and 30-min warm pulse. One hundred autoradiograms were scored for each datum. p These data are all significantly different from the appropriate controls, P < 0.001 for the indicated data in columns 3, P < 0.05 for the data in columns 4 and 5 (Student’s t test).
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from control cells. The heavy grain tracks represent DNA replicated during the hot pulse, and the lighter grain tracks, DNA replicated during the warm pulse. Clusters of replication units usually show patterns indicating synchronous initiation (7, 8). Figure la shows three adjacent units that initiated replication before the [“Hlthymidine pulse. Each of these three prepulse units (labeled p) has a clear space flanked by grain tracks of high density, then low density. The clear space represents DNA replicated before the pulse, the high and low density tracks, DNA replicated during the hot and warm pulses. Figure lb shows three adjacent units that initiated replication after the beginning of the hot pulse. Each of these three postpulse units (labeled q) has a central high density grain track representing replication during the hot pulse, flanked by low density grain tracks, representing replication during the warm pulse. In both la and b, the three units of the autoradiogram initiated replication at approximately the same time. Less frequently, autoradiograms show mixtures of pre- and postpulse units (Fig. 1~). Synchrony of initiation may be measured by determining how often two or more adjacent replication units on an autoradiogram show the same pattern (either all prepulse or all postpulse). Random initiation would be indicated by the frequency of each of the possible combinations of pre- and postpulse units approaching that predicted by the binomial equation (p + q)” = 1, where p equals the proportion of prepulse units q, the proportion of postpulse units, and n, the number of units per autoradiogram. In control cells, about 80% of 2-unit autoradiograms show like pairs whereas only 50% like pairs would be expected if initiation were random (8,121. Most replication units in control cells show bidirectional replication. Occasionally, internal units with unidirectional replication are seen (Fig. Id, u). When single internal units are scored, usually 80% or more are bidirectional (1 O-12). The hot pulse track lengths of prepulse units (labeled p in Fig. la-d) can be used to measure rate of replication fork progression. The beginning of the hot pulse is
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marked by the appearance of grains, the end by the transition from heavy to light grain density. Normally, the rate is 0.60.7 pm/min/fork (1800-2100 base pairs/ min/fork) (6, 9, II). Figure 2 shows autoradiograms of DNA from cells 6 hr after infection with mengovirus (Fig. 2a, b) or NDV (Fig. Zc, dl. The autoradiograms illustrated show mixtures of pre- and postpulse units indicating more random initiation. Unidirectional units are seen more frequently. The hot pulse track lengths also appear shorter indicating slower rates of fork progression. The data from the analyses of the autoradiograms from control and infected cells are presented in Table 1, columns 3-5. Rate of replication fork progression in L cells 5 hr after infection with either virus is normal, as shown previously (6). By 6 hr after infection, mengovirus infection has reduced fork progression by more than 60% and NDV infection has reduced it by close to 50% (column 3). Direction of replication and synchrony of initiation (Table 1, columns 4-5) reflect initation events. The normal values for these measurements in L cells have been reported (8, II, 12) and the control values in the present experiments are similar. At 5 hr after infection with mengovirus or NDV they are unchanged. By 6 hr after infection, the normal initation patterns have been altered in infected cells. The DNA of infected cells shows a decrease in the proportion of units with bidirectional replication (column 4). This indicates a change in normal function at the initiation site of active units, in that one fork, rather than two, results from an initiation event. The synchrony of initiation is also decreased in infected cells by 6 hr after infection (column 5) in that pairs of adjacent units in DNA are less frequently of the same type (either both prepulse or both postpulse). The same analysis was carried out on autoradiograms containing three units. A similar result was obtained in that DNA initiation was more random in infected cells (data not shown). The analysis could not be extended to four-unit autoradiograms, since there were insufficient numbers of these available in preparations from infected cells. The conclusion can still
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FIG. 1. DNA fiber autoradiograms from control cells. DNA was labeled with LJH]thymidine for a 30-min hot followed by a 30-min warm pulse. The arrows show the direction of a particular replication fork. p, Prepulse unit; q, postpulse unit; u, unidirectional unit. (a) Three tandem prep&e units. (b) Three tandem postpulse units. (c) A postpulse unit adjacent to two prep&e units. (d) A unidirectional unit flanked by two prepulse units. The bar in (a) indicates 100 pm. All micrographs are at the same magnification.
be drawn that synchrony of initiation is decreased in infected cells. The data show that by 6 hr after infection both mengovirus and NDV have altered the normal pattern of initiation of chromosomal DNA replication and have slowed the rate of replication fork progression. At 5 hr postinfection, there are no demonstrable changes in the autoradiogram patterns despite a significant decrease in thymidine incorporation into DNA of infected cells. There are several possible explanations for this. (9 Initiation of replication on whole clusters might be inhibited by the viruses. The residual replication visualized by autoradiography might be normal. (ii) The decrease in thymidine incorporation at 5 hr might be explained completely or in part by an inhibition of transport of exogenous thymidine resulting from membrane changes induced by virus infection. An apparent decrease in cellular RNA synthesis induced by vesicular stomatitis virus infection has been
attributed to a decrease in membrane transport of [3H]uridine (19). (iii) The decrease incorporation may not represent a true decrease in DNA synthesis, but rather nucleotide pool dilution by endogenous synthesis and synthesis of DNA of lower specific activity in infected cells. An increase in exogenous thymidine incorporation resulting from inhibition of DNA synthesis has been observed in Sendai virus-infectgd cells (20) and the drawbacks of relying completely on thymidine incorporation as an indicator of DNA synthesis have been pointed out (21). Experiments are in progress to determine the cause of decreased thymidine incorporation at the earlier times after infection. Both mengovirus and NDV inhibit L cell protein synthesis (1,5) and their effects on chromosomal DNA replication 6 hr after infection are similar to those produced by inhibition of cellular protein synthesis by cycloheximide and puromycin (12). Thus, as suggested previously (51, the cut-off of
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FIG. 2. DNA fiber autoradiograms from virus-infected cells. DNA was labeled with 13H1thymidine for a 30-min hot followed by a 30-min warm pulse. The midpoint of the hot pulse was 6 hr after infection. The arrows and symbols are the same as in Fig. 1. (a) DNA from mengovirus-infected cells. A long fiber is shown with three tandem unidirectional units at the top and adjacent postpulse and prepulse units toward the bottom. Another fiber with a unidirectional unit is immediately to the right of the long fiber. The unlabeled arrow marks the unit adjacent to the unidirectional unit, indicating the unidirectional unit is internal. (b) DNA from mengovirus-infected cells. A long fiber is shown containing (from top to bottom) a prepulse, unidirectional, postpulse, and prepulse unit. Immediately to the right is a fiber with two unidirectional units, and again to the right, another fiber with a unidirectional unit. The unlabeled arrows show the unidirectional units to be internal as in (a). (c) DNA from NDV-infected cells. A fiber containing (from top to bottom) a prepulse unit, two unidirectional units with forks moving in opposite directions, a postpulse and a prepulse unit. (d) DNA from NDV-infected cells. A fiber with two adjacent unidirectional units. The unlabeled arrows above and below show the unidirectional units to be internal. The bar in (a) represents 100 pm. All micrographs are at the same magnification
cellular protein synthesis probably causes the inhibition of DNA replication. In the productive mengovirus infection, early synthesis of a relatively small amount of virus-specific protein results in the inhibition of cellular protein synthesis (17). In the abortive NDV infection, the restrictive step occurs late in the cycle (22) and, if events to that point in the abortive cycle are similar to those in productive infection of chick cells, early viral RNA and protein synthesis is necessary for the inhibition of cellular protein synthesis (23). The more gradual onset of the inhibition of DNA replication in the virus-infected cells as compared to antibiotic-treated cells may
be related to the time necessary for accumulation of adequate amounts of the virus-specific inhibitors of protein synthesis. ACKNOWLEDGMENTS Mlles. Colette Oblin and Dominique Brunet provided technical assistance. The investigation was supported by Grants No. MA-5143 from the Medical Research Council of Canada and No. l-348 from The National Foundation, March of Dimes. REFERENCES 1. FRANKLIN, R. M., and BALTIMORE, D., Cold Spring Harbor Symp. Quant. Biol. 27,175-196 (1962). 2. WHEELOCK, E. F., and TAMM, I., J. Exp. Med. 114, 617-632 (1961). 3. ENSMINGER, W. D., and TAMM, I., J. Virol. 5,
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672-676 (1970). 4. HAND, R., and TAMM, I., d. Viral. 8, 597-600 (1971). 5. ENSMINGER, W. D., and TAMM, I., Virology 40, 152-165 (1970). 6. HAND, R., and TAMM, I., Virology 47, 331-337 (1972). 7. HAND, R., and TAMM, I., J. Mol. Biol. 82, 175183 (1974). 8. HAND, R., J. Cell Biol. 64, 89-97 (1975). 9. HUBERMAN, J. A., and RIGGS, A. D., J. Mol. Biol. 32, 327-341 (1968). 10. HUBERMAN, J. A., and ‘hAI, A., J. Mol. Biol. 75, 5-12, (19731. 11. HAND, R., and TAMM, I., J. Cell Biol. 58,410-418 (1973). 12. HAND, R., J. Cell Biol. 67, 761-773 (1975). 13. WEINTAUB, H., and HOLTZER, J., J. Mol. Biol. 66, 13-35 (1972).
14. GAUTSCHI, J. R., and KERN, R. M., Exp. Cell Res. 86, 15-27 (1973). 15. GAUTSCHI, J. R., J. Mol. Biol. 84,223-229 (1974). 16. AMAKO, K., and DALES, S., Virology 32, 181-200 (1967).
17. COLLINS, F. D., and ROBERTS, W. K., J. Virol. 10, 969-978 119721. 18. COLBY, D. S., FINNERTY, V., and LUCAS-LENARD, J., J. Viral. 13, 858-869 (1974). 19. GENTY, N., J. Virol. 15, 8-15 (1975). 20. FUCHS, P., and KOHN, A., J. Viral. 8, 695-700 (1971). 21. HAUSCHKA, P. V., Ztz “Methods in Cell Biology VII” CD. Prescott, ed.), pp. 362-462. Academic Press, New York (1973). 22. HECHT, T. T., and SUMMERS, D. F., J. Virol. 14, 162-169 (1974). 23. HIGHTOWER, L. E., and BRATT, M. A., J. Viral. 13, 788-800 (1974).
71, 609-614 (1976)
Chromosomal DNA Replication: Retarded Fork Progression and Altered Initiation in Cells Infected with Wlengovirus or Newcastle Disease Virus ROGER HAND Departments
of Medicine
and Microbiology,
McGill
University,
Accepted January
Montreal,
Quebec H3A 2B4, Canada
13,1976
Infection of mouse L cells with mengovirus or Newcastle disease virus inhibits DNA synthesis. The effects of this inhibition on chromosome replication have now been investigated using DNA fiber autoradiography, a technique permitting analysis of events on clusters of active replication units. At 5 hr after infection at high multiplicity with either virus, DNA replication is normal despite a marked inhibition of [3H]thymidine incorporation into DNA. By 6 hr, virus infection slows the rate of replication fork progression. In addition, virus infection alters the pattern of initiation of DNA replication in that the proportion of units with bidirectional replication is reduced and the usual synchrony of initiation events on clusters of units is decreased.
Infection of mouse L-929 cells productively with mengovirus, a picornavirus, or abortively with Newcastle disease virus (NDV), a paramyxovirus, results in the inhibition of incorporation of 13Hlthymidine into cellular DNA (l-3). Since neither virus alters the percentage of cells in the DNA synthetic phase of the cell cycle (4) or significantly slows the rate of replication fork progression (daughter chain elongation) (5, 61, both viruses are thought to block the multifocal initiation of DNA synthesis. The normal patterns of DNA initiation in mammalian cells have been described. Initiation occurs synchronously on clusters of replication units along the chromosome f7,8). Replication proceeds bidirectionally from the origins of these units, and the newly replicated chains eventually fuse with daughter chains from adjacent units (g-11). Inhibition of cellular protein synthesis with the antibiotics puromycin and cycloheximide alters these patterns (12). The frequency of initiation and the synchrony of initiation events on clusters is decreased and the proportion of units with bidirectional replication is reduced. In addition, these antibiotics inhibit repli-
cation fork progression (6, 11, 13-15). Since the effects of both mengovirus and NDV on DNA replication have been attributed to the concomitant inhibition of cellular protein synthesis (5, 6), it would be of interest to know whether these viruses alter initiation patterns in the same fashion as the antibiotics and under what circumstances, if any, they inhibit replication fork progression. In the present experiments, DNA fiber autoradiography, a technique that permits analysis of events on active replication units on the chromosome, has been used to analyze DNA replication patterns in infected cells. At 5 hr after infection, rate of replication fork progression, the incidence of bidirectional replication, and the synchrony of initiation in infected cells and controls are similar. By 6 hr after infection, all three parameters are decreased in infected cells. Monolayers of L-929 cells were infected with mengovirus, small plaque variant (16) at a m.o.i. of 200 PFU/cell or NDV, Hickman strain (2) at a m.o.i. of 500 PFU/ cell. The characteristics and the conditions for propagation of the viruses and cell lines have been described (5, 12). Adsorption was for 1 hr in one-tenth the usual amount 609
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of medium. Control cells were sham-infected. At 5 and 6 hr after infection, thymidine incorporation into DNA was determined in control and infected cells by pulse-labeling them with [3H]thymidine (Table 1, column 2). Thymidine incorporation into DNA is inhibited in infected cells. Under these experimental conditions, mengovirus-infected cells have normal morphology 5 hr after infection. By 6 hr after infection, cell rounding is present in lo-20% of the cells. NDV-infected cells show normal morphology through 7 hr after infection. [3H]leucine incorporation into proteins is decreased 6 hr after infection with either virus. Incorporation is 41.9% of controls in mengovirus-infected cells and 37.1% of controls in NDV-infected cells. The degree of inhibition of incorporation of thymidine and leucine into DNA and protein is comparable to that reported by others (1, 5, 17,18). Replicate monolayers of control and infected cells were pulse-labeled with TABLE DNA Experiment
1. 5-hr postinfection Control Mengo-infected NDV-infected 2. 6-hr postinfection Control Mengo-infected NDV-infected
13Hlthymidine and their DNA processed for fiber autoradiography (7,ll). This involved treatment of the cells with fluorodeoxyuridine (2 x 10e6M) for 30 min before the pulse to exhaust their endogenous thymidine nucleotide pools, and sequential labeling with 13Hlthymidine at high specific activity (50 Ci/mmole, 5 X lo+ M, the hot pulse) and low specific activity (5 Ci/mmole, 5.5 x lo-” M, the warm pulse) for 30 min each. Fluorodeoxyuridine remained in the medium during the pulses. The times postinfection indicated in Table 1, column 1, represent the midpoints of the hot pulse. At the completion of the pulses, the DNA was processed for fiber autoradiography by lysing the cells gently and spreading the DNA fibers on a glass slide. The preparations were dried and fixed, and then coated with light-sensitive emulsion. After 3-6 months exposure, the slides were developed and examined by light microscopy. Figure 1 shows several autoradiograms 1
REPLICATION IN VIRUS-INFECTED CELLS
PHlthymidine incor orationa (% 0 P control)
Rate of fork progression* (pm/min t SEM)
Bidirectional replication units, proportionc
Initiation synchrony (pairs of units with like patterns), proportiond
100.0 37.6 46.0
0.72 + 0.043 0.67 2 0.034 0.69 k 0.036
0.82 0.75 0.76
0.78 0.76 0.78
100.0 5.6 21.4
0.64 + 0.023 0.24 2 0.015 0.35 k 0.017’
0.78 0.65’ 0.64’
0.77 0.53’ 0.59
n Measured in replicate monolayers handled in an identical manner to control and experimental cells except that in lieu of undergoing the DNA fiber autoradiography protocol, the cells were pulse-labeled with [“Hlthymidine (0.5 &i/ml) for 10 min at 5 or 6 hr after infection. Radioactivity incorporated into DNA was determined by acid precipitation and liquid scintillation counting. b Determined by measuring hot pulse track lengths of prepulse units in DNA sequentially labeled during a 30-min hot and 30-min warm pulse. On such tracks, the beginning and end of the hot pulse are marked. The length is divided by the duration of the hot pulse to obtain micrometers per minute. Fifty tracks were measured for each datum. c Determined by scoring internal replication units as to whether they showed unidirectional or bidirectional replication. DNA was labeled during a 30-min hot and 30-min warm pulse. One hundred units were scored for each datum. d Determined by scoring two-unit autoradiograms as to whether they contained a like pair or unlike pair of replication units. DNA was labeled during a 30-min hot and 30-min warm pulse. One hundred autoradiograms were scored for each datum. p These data are all significantly different from the appropriate controls, P < 0.001 for the indicated data in columns 3, P < 0.05 for the data in columns 4 and 5 (Student’s t test).
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from control cells. The heavy grain tracks represent DNA replicated during the hot pulse, and the lighter grain tracks, DNA replicated during the warm pulse. Clusters of replication units usually show patterns indicating synchronous initiation (7, 8). Figure la shows three adjacent units that initiated replication before the [“Hlthymidine pulse. Each of these three prepulse units (labeled p) has a clear space flanked by grain tracks of high density, then low density. The clear space represents DNA replicated before the pulse, the high and low density tracks, DNA replicated during the hot and warm pulses. Figure lb shows three adjacent units that initiated replication after the beginning of the hot pulse. Each of these three postpulse units (labeled q) has a central high density grain track representing replication during the hot pulse, flanked by low density grain tracks, representing replication during the warm pulse. In both la and b, the three units of the autoradiogram initiated replication at approximately the same time. Less frequently, autoradiograms show mixtures of pre- and postpulse units (Fig. 1~). Synchrony of initiation may be measured by determining how often two or more adjacent replication units on an autoradiogram show the same pattern (either all prepulse or all postpulse). Random initiation would be indicated by the frequency of each of the possible combinations of pre- and postpulse units approaching that predicted by the binomial equation (p + q)” = 1, where p equals the proportion of prepulse units q, the proportion of postpulse units, and n, the number of units per autoradiogram. In control cells, about 80% of 2-unit autoradiograms show like pairs whereas only 50% like pairs would be expected if initiation were random (8,121. Most replication units in control cells show bidirectional replication. Occasionally, internal units with unidirectional replication are seen (Fig. Id, u). When single internal units are scored, usually 80% or more are bidirectional (1 O-12). The hot pulse track lengths of prepulse units (labeled p in Fig. la-d) can be used to measure rate of replication fork progression. The beginning of the hot pulse is
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marked by the appearance of grains, the end by the transition from heavy to light grain density. Normally, the rate is 0.60.7 pm/min/fork (1800-2100 base pairs/ min/fork) (6, 9, II). Figure 2 shows autoradiograms of DNA from cells 6 hr after infection with mengovirus (Fig. 2a, b) or NDV (Fig. Zc, dl. The autoradiograms illustrated show mixtures of pre- and postpulse units indicating more random initiation. Unidirectional units are seen more frequently. The hot pulse track lengths also appear shorter indicating slower rates of fork progression. The data from the analyses of the autoradiograms from control and infected cells are presented in Table 1, columns 3-5. Rate of replication fork progression in L cells 5 hr after infection with either virus is normal, as shown previously (6). By 6 hr after infection, mengovirus infection has reduced fork progression by more than 60% and NDV infection has reduced it by close to 50% (column 3). Direction of replication and synchrony of initiation (Table 1, columns 4-5) reflect initation events. The normal values for these measurements in L cells have been reported (8, II, 12) and the control values in the present experiments are similar. At 5 hr after infection with mengovirus or NDV they are unchanged. By 6 hr after infection, the normal initation patterns have been altered in infected cells. The DNA of infected cells shows a decrease in the proportion of units with bidirectional replication (column 4). This indicates a change in normal function at the initiation site of active units, in that one fork, rather than two, results from an initiation event. The synchrony of initiation is also decreased in infected cells by 6 hr after infection (column 5) in that pairs of adjacent units in DNA are less frequently of the same type (either both prepulse or both postpulse). The same analysis was carried out on autoradiograms containing three units. A similar result was obtained in that DNA initiation was more random in infected cells (data not shown). The analysis could not be extended to four-unit autoradiograms, since there were insufficient numbers of these available in preparations from infected cells. The conclusion can still
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FIG. 1. DNA fiber autoradiograms from control cells. DNA was labeled with LJH]thymidine for a 30-min hot followed by a 30-min warm pulse. The arrows show the direction of a particular replication fork. p, Prepulse unit; q, postpulse unit; u, unidirectional unit. (a) Three tandem prep&e units. (b) Three tandem postpulse units. (c) A postpulse unit adjacent to two prep&e units. (d) A unidirectional unit flanked by two prepulse units. The bar in (a) indicates 100 pm. All micrographs are at the same magnification.
be drawn that synchrony of initiation is decreased in infected cells. The data show that by 6 hr after infection both mengovirus and NDV have altered the normal pattern of initiation of chromosomal DNA replication and have slowed the rate of replication fork progression. At 5 hr postinfection, there are no demonstrable changes in the autoradiogram patterns despite a significant decrease in thymidine incorporation into DNA of infected cells. There are several possible explanations for this. (9 Initiation of replication on whole clusters might be inhibited by the viruses. The residual replication visualized by autoradiography might be normal. (ii) The decrease in thymidine incorporation at 5 hr might be explained completely or in part by an inhibition of transport of exogenous thymidine resulting from membrane changes induced by virus infection. An apparent decrease in cellular RNA synthesis induced by vesicular stomatitis virus infection has been
attributed to a decrease in membrane transport of [3H]uridine (19). (iii) The decrease incorporation may not represent a true decrease in DNA synthesis, but rather nucleotide pool dilution by endogenous synthesis and synthesis of DNA of lower specific activity in infected cells. An increase in exogenous thymidine incorporation resulting from inhibition of DNA synthesis has been observed in Sendai virus-infectgd cells (20) and the drawbacks of relying completely on thymidine incorporation as an indicator of DNA synthesis have been pointed out (21). Experiments are in progress to determine the cause of decreased thymidine incorporation at the earlier times after infection. Both mengovirus and NDV inhibit L cell protein synthesis (1,5) and their effects on chromosomal DNA replication 6 hr after infection are similar to those produced by inhibition of cellular protein synthesis by cycloheximide and puromycin (12). Thus, as suggested previously (51, the cut-off of
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FIG. 2. DNA fiber autoradiograms from virus-infected cells. DNA was labeled with 13H1thymidine for a 30-min hot followed by a 30-min warm pulse. The midpoint of the hot pulse was 6 hr after infection. The arrows and symbols are the same as in Fig. 1. (a) DNA from mengovirus-infected cells. A long fiber is shown with three tandem unidirectional units at the top and adjacent postpulse and prepulse units toward the bottom. Another fiber with a unidirectional unit is immediately to the right of the long fiber. The unlabeled arrow marks the unit adjacent to the unidirectional unit, indicating the unidirectional unit is internal. (b) DNA from mengovirus-infected cells. A long fiber is shown containing (from top to bottom) a prepulse, unidirectional, postpulse, and prepulse unit. Immediately to the right is a fiber with two unidirectional units, and again to the right, another fiber with a unidirectional unit. The unlabeled arrows show the unidirectional units to be internal as in (a). (c) DNA from NDV-infected cells. A fiber containing (from top to bottom) a prepulse unit, two unidirectional units with forks moving in opposite directions, a postpulse and a prepulse unit. (d) DNA from NDV-infected cells. A fiber with two adjacent unidirectional units. The unlabeled arrows above and below show the unidirectional units to be internal. The bar in (a) represents 100 pm. All micrographs are at the same magnification
cellular protein synthesis probably causes the inhibition of DNA replication. In the productive mengovirus infection, early synthesis of a relatively small amount of virus-specific protein results in the inhibition of cellular protein synthesis (17). In the abortive NDV infection, the restrictive step occurs late in the cycle (22) and, if events to that point in the abortive cycle are similar to those in productive infection of chick cells, early viral RNA and protein synthesis is necessary for the inhibition of cellular protein synthesis (23). The more gradual onset of the inhibition of DNA replication in the virus-infected cells as compared to antibiotic-treated cells may
be related to the time necessary for accumulation of adequate amounts of the virus-specific inhibitors of protein synthesis. ACKNOWLEDGMENTS Mlles. Colette Oblin and Dominique Brunet provided technical assistance. The investigation was supported by Grants No. MA-5143 from the Medical Research Council of Canada and No. l-348 from The National Foundation, March of Dimes. REFERENCES 1. FRANKLIN, R. M., and BALTIMORE, D., Cold Spring Harbor Symp. Quant. Biol. 27,175-196 (1962). 2. WHEELOCK, E. F., and TAMM, I., J. Exp. Med. 114, 617-632 (1961). 3. ENSMINGER, W. D., and TAMM, I., J. Virol. 5,
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