J. gen. Virol. (1983), 64, 1991-1998. Printed in Great Britain
1991
Key words: MVM/parvovirus/DNA synthesis/aphidicolin
Interrelation between Viral and Cellular DNA Synthesis in Mouse Cells
Infected with the Parvovirus Minute Virus of Mice
By N. H A R D T , 1 C. D I N S A R T , z S. S P A D A R I , 2
G. P E D R A L I - N O Y 2 AND J. R O M M E L A E R E ~*
1Laboratoire de Biophysique et Radiobiologie, Universit~ libre de Bruxelles, rue des Chevaux, 67,
B-1640 Rhode-St Genkse, Belgium and Zlstituto di Genetica Biochimica ed Evoluzionistica,
Consiglio Nazionale delle Ricerche, Via S. Epifanio, 14, 1-27100 Pavia, Italy
(Accepted 25 May 1983)
SUMMARY
Mouse fibroblasts arrested in Go by isoleucine deprivation were inoculated with the
autonomous parvovirus minute virus of mice (MVM). Infected cells were released from
the Go block by transfer to complete medium and their progression to and through the S
phase was monitored. The onset of viral and cellular DNA synthesis coincided, suggesting that cellular factor(s) required for MVM DNA replication became available as
soon as cells entered the S phase. Cellular DNA synthesis was reduced to about 6 0 ~ by
MVM infection. However, this inhibition did not decrease significantly the overall rate
of DNA replication in infected cells because it was compensated by concomitant viral
DNA synthesis. MVM infection delayed the movement of the cells out of S phase by at
least 5 h. At any time post-infection, more than 9 5 ~ of both viral and cellular DNA
synthesis was sensitive to inhibition by aphidicolin. Since this drug is highly specific for
cellular DNA polymerase ~, the data are consistent with a major role of this enzyme in
the in vivo DNA replication of autonomous parvovirus. The assembly of 95 ~ of virus
progeny particles was concomitant with a late phase of viral DNA replication which
accounted for 30 ~ of the total viral DNA synthesized. The inhibition of this residual
viral DNA replication by aphidicolin reduced dramatically the size of the burst of
infectious particles; this observation concurs with other evidence to suggest that
encapsidation is driven by a late replication event sensitive to this drug.
INTRODUCTION
Parvoviruses are among the smallest known animal viruses and contain a predominantly
single-stranded, linear DNA genome (Tattersall & Ward, 1978). The replication of parvovirus
DNA takes place in the cell nucleus and involves three steps: (i) the conversion of singlestranded DNA to double-stranded replicative forms, (ii) the replication of double-stranded
DNA and (iii) the asymmetric production of progeny single-stranded DNA from replicative
forms (Berns & Hauswirth, 1978). Parvoviruses of the non-defective subgroup are able to replicate
autonomously provided that S phase-specific host cell function(s) is (are) available (Tattersall &
Ward, 1978). The S phase-dependent step of the parvovirus life cycle is likely to be viral DNA
replication, in particular the conversion of the input single-stranded genome to a duplex form
(Wolter et al., 1980). The cellular 'S factors' essential for parvovirus replication have not been
identified so far and the time of their appearance during the S phase is disputed (Parris & Bates,
1976; Wolter et al., 1980). Productive infection by the non-defective parvoviruses does not
require a virion-associated DNA polymerase (Pritchard et al., 1978a) and is likely to rely on
cellular DNA polymerases. Although both DNA polymerases ~ and ~ have been shown to
catalyse parvovirus DNA replication in vitro (Handa & Carter, 1979; Pritchard et al., 1981;
Kollek & Goulian, 1981; Kollek et al., 1982; Faust & Rankin, 1982), the contribution of these
enzymes to viral DNA synthesis in vivo is unclear. The involvement of DNA polymerase ~ is
supported by indirect evidence showing a temporal correlation between this enzymic activity
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0022-1317/83/0000-5661 $02.00 © 1983 SGM
1992
N.
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and parvovirus D N A synthesis in infected cells (Pritchard et al., 1978 b). Studies of the effect of
parvovirus replication on the progression of cellular D N A replication have given contradictory
results. Some authors have reported that parvovirus infection causes an early reduction in the
rate of D N A synthesis (Salzman et al., 1972) whereas others have found no alteration in the progression of infected cells through S phase (Hampton, 1970; Parris & Bates, 1976).
These uncertainties concerning the interrelation between cellular and parvovirus D N A
replication prompted us to investigate the time course of these processes in synchronized mouse
cells following infection with the autonomous parvovirus minute virus of mice (MVM) (Ward &
Tattersall, 1982). The data presented suggest that MVM D N A replication (i) depends on cellular
function(s) expressed early in S phase, (ii) is accompanied by an inhibition of cellular D N A
synthesis, and (iii) is inhibited by aphidicolin, a specific inhibitor of cellular D N A polymerase
(Ikegami et al., 1978; Pedrali-Noy & Spadari, 1979), to a similar extent as cellular D N A replication. Moreover, a late, aphidicolin-sensitive event in MVM D N A replication seems to drive
the assembly of infectious progeny particles.
METHODS
Cell culture and viral infection. Mouse A9 cells (Littlefield, 1964) were accumulated in G O by isoleucine deprivation as described previously (Rommelaere et al., 1981). The arrested monolayer culture (4 × 105 cells) was
infected with the plaque-purified strain T of MVM (Tattersall, 1972) at 4 p.f,u./cell. Immediately after infection,
cells were transferred to Eagle's minimal essential medium supplemented with 5 ~ foetal calf serum.
Autoradiography. At intervals following MVM or mock infection, cells attached to glass slides were pulselabelled with [3H]thymidine (TdR) (5 ~tCi/ml, 5 Ci/mmol) for 45 min and further incubated for 10 min in phosphate-buffered saline (PBS) containing 1 mg/ml of unlabelled TdR. Cultures were then rinsed, fixed and prepared
for autoradiography, as described previously (Hardt et al., 1980).
Virus titration. At intervals post-infection, cells were collected and the virus was extracted by freeze-thawing
(Tattersall et al., 1976). Extracts were assayed for infectivity by direct plaque assay (Tattersall, 1972). In some
experiments, infected cells were exposed to 15 ktm-aphidicolin from diffferent times until 36 h post-infection, at
which time the infectivity of cell-associated viral particles was measured.
DNA analysis
Radioactive labelling. At intervals after MVM or mock infection, cells were pulse-labelled with [3H]TdR (20
pCi/ml, 25 Ci/mmol) for 45 min. Cultures were then incubated for 7 min with PBS containing l mg/ml of unlabelled thymidine. After several rinses, cells were collected and suspended in PBS (2 x 105 cells/ml). When present,
aphidicolin was used at 15 ~tM during both the pulse and chase periods.
Measurement o f the overall rate o f DNA synthesis. A 0.1 ml amount of the cell suspension was collected on a
Whatman GF/C filter. The filter was washed twice in 5~o trichloroacetic acid (TCA) and once in ethanol. When
dry, the filter was dipped in Econofluor scintillator (New England Nuclear) and the radioactivity was measured in
a liquid scintillation spectrometer. It was verified that the rate of incorporation of[3H]TdR in both uninfected and
MVM-infected cells was not changed by increasing the concentration of thymidine in the external medium from
0.8 ktM (our standard radioactive labelling conditions) to 5 pM. Therefore, the incorporation of [3H]TdR is not
likely to be affected by endogenous deoxyribonucleotide pools (Cleaver & Holford, 1965) and should provide a
reliable measurement of DNA synthesis.
Alkaline sucrose sedimentation analysis. The cell suspension was exposed to 20 Gy of y-radiation delivered from a
Gamma cell 200 (6°Co). A 4 ml gradient of 5 to 20~ sucrose in 0.1 M-NaC1, 0.1 M-NaOH was formed on top of a 1
ml cushion of 50~ sucrose. Cell suspensions were made 0.2 Min NaOH, incubated for 15 rain at room temperature
and layered (0-2 ml) on top of the gradients. These were centrifuged immediately at 4 °C, in the SW50.1 rotor in a
Beckman model L ultracentrifuge at 48000 rev/min for 4 h. After centrifugation, 24 to 26 fractions were collected
and their radioactivity was measured by liquid scintillation spectrometry using Aquasol-2 fluid (New England
Nuclear). DNA purified from MVM virions and labelled with [3H]TdR (Bourguignon et al., 1976) was used as a
marker and centrifuged in parallel gradients.
Determination ofL,iralDNA by D N A - D N A reassociation. Samples of labelled cells were suspended in 0.02 M-Tris
HC1, 0-15 M-NaCI, 0.01 M-EDTA, pH 7-5 (2 × l07 cells/ml) and lysed with SDS (0.6%) and self-digested Pronase
(2 mg/ml) for 1 h at 37 °C. Extracted DNA was sheared to a length of 500 to 600 nucleotides by heating the lysates
at 100 °C in the presence of 0.3 M-NaOH for 15 min~ After cooling to 0 °C, samples were neutralized with K H , P O 4
to a final pH 7.3 to 7.5 and dialysed overnight against 5 mM-Tris-HC1, 0.5 M-NaCI, 1 mM-EDTA, pH 7.7. The
resultant DNA solutions were used for reassociation experiments as described by Pedrali-Noy & Weissbach (1977)
except that an appropriate excess of both strands of non-radioactive viral DNA was added to each sample to drive
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M V M D N A synthesis
1993
the reassociationreaction. This DNA was prepared by copyingwith Escherichia cob DNA polymerase I (Klenow
fragment) self-primed single-stranded MVM DNA purified from viral particles, essentially as described by
Bourguignon et al. (1976).
Chemicals. [Me-3H]TdR was from Amersham International. Aphidicolin was kindly supplied by Dr A. Todd,
Imperial Chemical Industries, Macclesfield, Cheshire, U.K.
RESULTS
Pattern of overall D N A synthesis in MVM-infected cells
Mouse cells were accumulated in Go by incubation in isoleucine-depleted medium. Arrested
cultures were transferred to complete medium and pulse-labelled with [3H]TdR at intervals.
D N A synthesis was measured by determining the radioactivity incorporated into TCAinsoluble material and by cell autoradiography. Cultures released from the Go block resumed the
cell cycle in a synchronous fashion. Fig. 1 (solid lines) illustrates the time course of cellular D N A
synthesis. The traverse of S phase is indicated by a sharp and transient increase of both the
fraction of labelled cells (Fig. 1a) and the incorporated radioactivity (Fig. 1 b). Entry into S
phase and maximum labelling occurred about 10 and 18 h following transfer to complete
medium, respectively.
G0-arrested cultures were infected with MVM at a multiplicity of 4 infectious units/cell,
immediately prior to transfer to complete medium. MVM infection did not disturb significantly
the entry of the cells into S phase (Fig. 1 a). In contrast, the labelling index increased for 5 h more
in MVM- than in mock-infected cultures, indicating that the movement of MVM-inoculated
cells out of the phase of D N A synthesis was delayed (Fig. 1a). As illustrated by Fig. 1 (b), MVM
infection caused little reduction in the maximum rate of D N A synthesis but slowed its decline,
resulting in a 1.2-fold increase of the total amount of labelled D N A precursors incorporated
within 35 h post-infection.
Time course of viral and cellular D N A replication
Incorporation of [3H]TdR into cellular and viral D N A was measured in cells pulse-labelled at
intervals after Go release and MVM infection. The D N A isolated from infected cells was
fractionated by sedimentation through alkaline sucrose gradients; it generated bimodal profiles
(Fig. 2a). Molecules of high and low sedimentation rates co-migrated with D N A from
uninfected cells and with purified viral DNA, respectively. The slow-sedimenting peak was
used for the quantification of viral D N A (Fig. 2a). The fraction of virus-specific tritiated D N A
was also determined by D N A - D N A reassociation in the presence of an excess of non-radioactive viral D N A (Table 1).
Although Fig. 1 (b) shows that the overall rate of D N A synthesis in infected cells closely
approximated that of uninfected cells, Fig. 2 (b) clearly shows that cellular D N A replication was
markedly depressed after MVM infection. However, this inhibition, presumably due to some
event(s) in MVM replication, was compensated for by a concomitant increase in viral D N A synthesis. The extent of the inhibition of cellular D N A synthesis occurring within 35 h after
infection varied from 20 to 6 0 ~ in independent experiments. Correspondingly, viral D N A
replication accounted for a fraction, which ranged from 25 to 65 %oof total incorporated radioactivity. The reason for this fluctuation in the level of viral D N A synthesis is not known. The
time courses of viral and cellular D N A synthesis in infected cultures overlapped to a great extent
(Fig. 2b; Table 1). The onset of replication of both types of D N A could not be dissociated and
took place around 10 h after infection and release from Go. Infected cells achieved the highest
rate of viral D N A replication about 5 h after residual cellular D N A synthesis started to decline.
Effect of aphidicolin on viral D N A replication and encapsidation
Viral replication was tested for its sensitivity to aphidicolin. The drug was used at a concentration, 15 gM, that inhibits over 95~o of cellular D N A replication (Pedrali-Noy & Spadari,
1980) with no effect on R N A and protein synthesis (Pedrali-Noy et al., 1982). Consistently,
aphidicolin reduced [3H]TdR incorporation into cellular D N A of both mock- and MVMDownloaded from www.microbiologyresearch.org by
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1994
N.
HARDT
AND
OTHERS
I
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Fig. 2
Fig. 1. Effect of MVM infection on overall DNA synthesis in synchronized A9 ceils. Cultures were
pulse-labelled with [3H]TdR at intervals following release from G Oblock and MVM infection. (a) Percentage of labelled cells determined by autoradiography. At least 3000 cells were examined for each
point. Similar results were obtained with autoradiograms exposed from 6 h up to 20 days. (b)
Radioactivity incorporated into TCA-insoluble material. Cells were pulse-labelled in the absence (O,
A) or presence ( 0 , A) of aphidicolin. O, O, Uninfected cells; A, A , MVM-infected cells.
Fig. 2. Time course and aphidicolin sensitivity of cellular and viral DNA synthesis in MVM-infected
A9 cells. A9 cells were pulse-labelled with [3H]TdR at intervals after MVM or mock infection and
release from Go block. (a) DNA fractionation by alkaline sucrose gradient sedimentation. Lysates of
MVM-infected (O) and mock-infected (0) cells harvested 24 h after infection were run in parallel
gradients. The arrow indicates the position of purified MVM virion DNA. For the purpose of quantification, DNA within the region of the gradient defined by the bar was considered to be of viral origin. (b)
Rates of viral and cellular DNA synthesis. Cultures were pulse-labelled in the absence (O, A , IS]) or
presence (O, A, II) of aphidicolin. The radioactivity incorporated into viral and cellular DNA
fractions (see a) was measured at different times post-infection. O, 0 , Cellular DNA from mockinfected cells; A, A , cellular DNA from MVM-infected cells; I-q, II, viral DNA from MVM-infected
cells.
infected A9 cells to 3 or 5 ~ of its n o r m a l level at any t i m e after release f r o m Go (Fig. 1 b a n d 2 b).
M o r e o v e r , viral D N A in M V M - i n f e c t e d cells was as sensitive to a p h i d i c o l i n as cellular D N A
replication, irrespective o f t i m e post-infection (Fig. 2b). A p h i d i c o l i n did not alter the f r a c t i o n o f
the culture i n v o l v e d in D N A synthesis but rather the level o f i n c o r p o r a t i o n p e r r e p l i c a t i n g cell
(data not shown). T h e influence of the i n h i b i t i o n o f D N A synthesis on the f o r m a t i o n o f
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M V M DNA synthesis
1995
T a b l e 1. Time course of viral and cellular DNA synthesis in MVM-infected A9 cells
Rate of DNA synthesis (ct/min/104 cells)*
Time post-infection (h)
5
19
26
32
45
Total DNA t
2050
27800
28679
6219
5720
Viral DNA~
ND[]
3367
6564
1293
795
Cellular DNA§
ND
24433
22115
4926
4925
* Cultures were pulse-labelled with [3H]TdR and lysed at intervals following release from G O block and
infection. Radioactivity incorporated into TCA-insoluble material was measured.
t Total DNA extracted' from infected cells.
Virus-specific DNA, as determined by DNA DNA reassociation. The reassociation reaction was completed
within 8 min in the presence of 22 Ixg/ml of non-radioactive viral DNA. Values were corrected for the extent of
reassociation of i:ellular DNA under those conditions (approximately 5 ~).
§ Total DNA synthesis minus viral DNA synthesis.
II ND, Not done.
I
I
I
-3
f
x
<
Z
2~
0.5
. ~0
0
0
10
20
30
Time post-infection (h)
Fig. 3. Effect of aphidicolin on progeny virus formation in MVM-infected A9 cells, At intervals after
MVM infection, A9 cells were either collected (closed symbols, continuous line) or further incubated in
the presence of 15 ~tM-aphidicolinuntil harvest at 36 h post-infection (same open symbols, interrupted
lines). Collected cells were disrupted and the infectivity of associated viral particles was measured. The
dotted line is the cumulative curve of viral DNA synthesis calculated from Fig. 2(b) and considered as
1.0 at 36 h post-infection.
infectious progeny particles was studied in M V M - i n f e c t e d cultures exposed to a p h i d i c o l i n from
different times to harvest time at 36 h after infection. It is a p p a r e n t from Fig. 3 that little virus
p r o d u c t i o n occurred in the presence of aphidicolin. I n the a b s e n c e of the drug, most of the viral
p r o g e n y was assembled d u r i n g the interval from 24 to 36 h post-infection. T h e p r o d u c t i o n of the
burst was c o n c o m i t a n t with a late phase o f viral D N A replication a c c o u n t i n g for only 30 ~ o f the
total viral D N A synthesized from the time of infection. T h e i n h i b i t i o n of this residual D N A
synthesis by aphidicolin (Fig. 2b) was associated with a d r a m a t i c r e d u c t i o n of the burst size
(Fig. 3). Thus, although a major fraction of total viral D N A had b e e n synthesized by 24 h, little
p r o g e n y single-stranded D N A was available for e n c a p s i d a t i o n unless a n a p h i d i c o l i n - s e n s i t i v e
event(s) occurred d u r i n g the s u b s e q u e n t 12 h.
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N. HARDT AND OTHERS
DISCUSSION
At any time after infection, aphidicolin reduced viral DNA synthesis by more than 9 5 ~ (Fig.
2b). Parvovirus DNA replication involves the conversion of input single-strands into duplex
parental replicative forms (RF). The multistep amplification of parental RF DNA gives rise to a
large pool of RF molecules and eventually to progeny single-stranded DNA (Berns &
Hauswirth, 1978). Parvovirus replication accounts for up to 75~ of uninfected cell DNA
synthesis within a 35 h period which comprises the crossing of S phase (Fig. 2b). Most of the
incorporation of [3H]TdR into viral DNA is associated with the viral DNA amplification
phase; the conversion of the few 5000 nucleotides-long input gcnomes contributes minimally to
total viral DNA synthesis. Consequently, the results presented indicate that aphidicolin prevents most of the amplification of parvovirus DNA, but they provide no information on the drug
sensitivity of parental RF formation.
Aphidicolin is known to inhibit cellular nuclear DNA replication (Geuskens et al., 1981). The
inhibition of viral DNA synthesis by this drug is unlikely to result merely from the inhibition of
cellular DNA replication since viral DNA synthesis does not require concomitant cellular D N A
replication (Parris & Bates, 1976). A more likely interpretation is that an aphidicolin-sensitivc
effector participates in a step of MVM DNA replication which is limiting for the amplification
of viral DNA throughout the virus life cycle.
The dependence of parvovirus DNA replication on host cell enzymes (Rhode, 1978) and the
specificity of aphidicolin for cellular DNA polymerase czstrongly suggest that the latter enzyme
is involved in viral DNA synthesis. It cannot be ruled out, however, that the drug affects other as
yet undiscovered cellular or viral factors involved in parvovirus DNA synthesis. The role of
DNA polymerase ct in DNA replication of autonomous parvovirus is further supported by the
temporal correlation between this enzymic activity and viral DNA synthesis in vivo (Pritchard et
al., 1978b) and by the ability of partially purified preparations of this enzyme to catalyse steps
of viral DNA synthesis in vitro (Pritchard et al., 1981 ; Kollek et al., 1982; Faust & Rankin, 1982;
Hfibscher et al., 1982; M. GiJnther, personal communication).
The inhibition of MVM DNA synthesis was greater than 95 ~ and not significantly different
from that of cellular DNA synthesis (Fig. 2 b). Such a sensitivity to aphidicolin would be consistent with the fact that amplification of the DNA of autonomous parvoviruses relies essentially or
even exclusively on DNA polymerase ct, as does the replication of cellular chromosomal DNA.
However, the DNA amplification of autonomous parvoviruses is a multistep process involving
the replication of RF molecules and the asymmetric production of single-stranded DNA of viral
polarity (Berns & Hauswirth, 1978). Thc temporal sequence and the enzymology of these steps
are still unclear. The permanent need for DNA polymerase ct therefore does not rule out
additional polymerase requirements for the completion of viral DNA synthesis. Such a
possibility is raised by recent studies showing that DNA polymerase ), participates in parvovirus
DNA replication in vitro (Kollek et al., 1982; M. Giinther, personal communication).
Very little new virus assembly occurrcd in the presence of aphidicolin (Fig. 3). Although a
major fraction of viral DNA had been synthesized by 24 h after infection, cells did not contain,
at this stage, a large pool of progeny viral strands which could be cncapsidated in the absence of
an aphidicolin-sensitive event. The specificity of this drug points to the dependence of MVM
production on DNA synthesis. The inhibition by aphidicolin of residual cellular D N A
replication is unlikely to account for the reduced formation of infectious particles since the
assembly and processing of autonomous parvoviruses do not require the host cell to be in S phase
(Parris & Bates, 1976; Richards et al., 1978). It follows that the burst of virus encapsidation
appears to require concomitant synthesis of viral DNA. A similar conclusion was drawn from
the effect of hydroxyurea on MVM production by rat cells (Richards et al., 1977, 1978). Little, if
any, free single-stranded DNA was found in infected cells (Tattcrsall & Ward, 1976), suggesting
that the production of progeny strands is tightly coupled to their packaging. The ultimate step in
the formation of viral single strands is thought to be their displacement from duplex forms by
asymmetric replication (Berns & Hauswirth, 1978). It was proposed that this strand displacemcnt synthesis may be driven by encapsidation (Tattersall & Ward, 1976; Richards et al., 1978)
thereby accounting for the dependence of the assembly of new particles on continuing viral
DNA synthesis.
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M V M D N A synthesis
1997
The burst of MVM packaging was associated with a late phase of viral DNA replication
accounting for only 30 ~ of the total viral DNA synthesized after infection (Fig. 3). Since virion
morphogenesis lags behind the amplification of the pool of RF DNA (Ward & Tattersall, 1982),
this late phase is likely to be mainly devoted to the asymmetric strand displacement synthesis of
single-stranded progeny DNA. The aphidicolin sensitivity of both virion assembly and late viral
DNA replication strongly suggests that DNA polymerase a participates in vivo in the displacement synthesis. This conclusion is consistent with the aphidicolin sensitivity of this reaction in
parvovirus DNA replication in vitro (Pritchard et al., 1981 ; Kollek et al., 1982).
A9 cells were accumulated in Go, inoculated with MVM and released from growth inhibition.
MVM uptake was presumably completed when cells entered into S phase since this process is
independent of the cell cycle and is carried out within a few hours after infection (Linser et al.,
1979; J. Rommelaere, unpublished results). Therefore, initiation of viral DNA replication was
mainly limited by the dependence of the conversion of the parental genome into duplex RF, on
cellular factors expressed specifically during the S phase ('S factors') (Tattersall, 1972). The
onset of viral DNA synthesis was found to coincide with the burst of cellular DNA replication
(Fig. 2b). Consistently, the first viral double-stranded DNA was detected in early S phase
(Wolter et al., 1980). In contrast, other authors reported that viral DNA synthesis was delayed
until the end of the S phase (Parris & Bates, 1976). Taken together, these data suggest that
cellular S factor(s) might be available throughout the S phase and that the relative timing of
cellular and viral DNA synthesis may vary depending on the system and cell synchronization
method used.
Viral DNA synthesis was accompanied by an inhibition of cellular DNA replication detectable as soon as 10 h after infection (Fig. 2b). Viral DNA synthesis might compete with cellular
DNA replication since it appears to make use, in particular, of the cellular DNA polymerase ct
(see above) and it accounts for as much as 75 ~ of thymidine incorporation during a normal S
phase (Fig. 2b; Parris & Bates, 1976). The decrease in RNA and protein syntheses (Parris &
Bates, 1976) and the degeneration of nuclear elements (Richards et al., 1977) which occur at the
time of viral DNA synthesis and virion assembly, respectively, might also participate in the
reduction of cellular DNA replication. The inhibition of cellular DNA synthesis was compensated by concomitant viral DNA replication. Therefore, MVM infection caused only a slight
decrease of the rate of overall thymidine incorporation, which was restricted to early S (Fig. 1 b).
Similarly, cells inoculated with the bovine (Parris & Bates, 1976) and hamster osteolytic
(Hampton, 1970) parvoviruses proceed through S phase without a significant reduction in the
rate of total DNA synthesis.
Taken together, the data presented and discussed point to the intimate relationship between
DNA replication of autonomous parvoviruses and their host cells with regard to their nuclear
location, cell cycle dependency and enzymology. In this respect, parvoviruses appear to provide
an attractive model for studying the molecular mechanism of DNA replication in eukaryotic
cells.
We wish to thank Professor M. Errera for his support and Drs J. Tal and J. J. Cornelis for helpful discussions.
This work was supported by an agreement between the Belgian Government and the Universit6 libre de Bruxelles
'Actions de Recherche Concert6e', the European Communities [contract BIO-359-B(G) and ENV/335/B], the
Minist6re de la Sant6 Publique (contract G/826/1981), the Fonds National de la Recherche M6dicale (contract
3.4514.80) and partly by the 'Programma finalizzato Controllo delle Malattie da Infezione'. J. Rommelaere is
Chercheur Qualifi6 du Fonds National Beige de la Recherche Scientifique.
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(Received 7 March 1983)
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