429
J. gen. ViroI. (I979), 42, 429-433
Printed in Great Britain
Studies on Polyoma Virus D N A Replication
in Synchronized C3H2K Cells
(Accepted z 5 September I978)
SUMMARY
In GI-arrested cells infected between I and 12 h after having been stimulated by
fresh serum to progress to S phase, polyoma virus DNA synthesis proceeded in the
first half of S phase, and virus and whole cellular DNA accumulated at about
the same time. However, in cells infected later than 14 h after serum stimulation,
virus DNA synthesis was shifted to the next S phase. Thus, a permissive cell
attains competence for polyoma virus DNA replication at a precise moment during
an S phase initiated by fresh serum, which can efficiently replace the early virus
host DNA stimulation function. When cells were incubated in serum that had
lost its capacity to stimulate host DNA synthesis by pre-absorption with growing
cells, normal yields of polyoma DNA could nevertheless be observed, which shows
that extensive replication of host DNA does not seem to be an obligatory condition
for virus DNA replication.
Synthesis of nuclear DNA of eukaryotes is a highly ordered but still poorly understood
process (Prescott, I976). The small and well-defined genomes of some of the DNA viruses,
which replicate their DNA during the S phase of their host cells, may be suitable probes to
study regulated DNA synthesis in eukaryotes.
Parvovirus H-I a single stranded DNA virus, replicates its DNA at the end of the S
phase (Rhode, I973) and it is known that different populations of eukaryotic DNA replicate at different times during the S phase (Balazs et al. I973)- The cellular double stranded
genomes of polyoma and SV4o viruses also replicate during the S phase (Manteuil et al.
I973; Thorne, I973 a, b; Pages et al. I975). For the latter viruses, however, a detailed study
of the relationship between virus and cellular DNA synthesis has not so far been made.
Thus, observations recorded for polyoma virus were based on virion formation and not
on the synthesis of virus DNA. Furthermore, the use of the metaphase detachment and the
double excess thymidine procedure which were used in these experiments did not allow
cells arrested in GI to be obtained in a reproducible way. This goal was achieved in the
present work using the permissive C3H2K mouse cell, a continuous cell line derived from
a mouse kidney fibroblast culture which, in previous work, has been found to be useful
for cell synchronization studies (Yoshikura et al. ~967). These cells have properties similar
to those of the 3T3 cell line (Todaro & Green, I965).
When grown to confluency or when kept in the same medium which became deficient,
C3H2K cells were arrested in GI phase. After renewal of nutrient medium, the cells progressed to S phase and divided in a synchronous way as determined by 3H-thymidine pulselabelling and cell counts. After a change to fresh medium, incorporation of radioactivity
was temporarily diminished in accordance with the work of Cramer & Feinendegen 0966).
A synchronized cycle of DNA replication started about I4 h after medium change and
maximum DNA synthesis was observed at 24 to 26 h. At this time, about 50 % of the cells
were found to be in the S phase as detected by autoradiography (not shown), confirming
oo22-I317/79/o0oo-2968 $o2.oo © 1979 SGM
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430
Table I. Time relationship between onset of S phase and virus DNA synthesis*
Time lapse between S phase and DNA
synthesis t in cultures infected
Time of infection
after medium change
(h)
c
Before medium
change
After medium
change
6
7
Io
6
4
9
6
5
8
7
5
6
8
5
8
9
6
9
Io
5
8
12
5
8
14
6
32
i6
6
30
i8
5
30
20
5
36
* In each experiment, GI arrested cells were infected before and at different times after medium change
and the kinetics of whole cellular and virus DNA synthesis were examined. For pulse labelling of cellular
DNA, cells were incubated at different times with I #Ci/ml 3H-thymidine(to mCi/mmol) for I h and the
synthesis was measured by the incorporation of radioactivity in the acid-precipitable fraction. To determine
its synthesis, the virus DNA was extracted by the Hirt 0967) method and assayed on mouse cultures for
infectivity.
t Values given are the time intervals in hours between the point at which the rate of thymidine incorporation into whole cellular DNA attained 5o% of maximal incorporation and the point at which half the yield
of virus was produced.
results already described for this cell system (Yoshikura & Hirokawa, I968). Forty-eight
hours after medium change, the cell number had doubled and a second, but usually incomplete, S phase was observed, followed by a progressive loss of synchronization. In
non-medium-changed cultures, plateau levels of D N A were observed.
This cell system allowed us to infect cells arrested in GI at different times after serum
stimulation and to study the time relationship of virus and cellular D N A synthesis (Table 0The length of the S period was determined by measuring radioactivity in the acid-precipitable material derived from synchronized pulse-labelled cells.
Continuous cell labelling was used for a comparative study of virus and cellular D N A
synthesis. Whole cell D N A synthesis was again examined as described. Virus D N A synthesis was determined by D N A infectivity titrations using a plaque-purified virus for which
I infectious unit of virus D N A was found to correspond to about Ios molecules of virus
D N A , comparable to earlier findings (Crawford, I969). Simultaneously, virus D N A synthesis was also determined by isolating virus D N A form I on equilibrium density gradients
(Radloff et al. I967).
Both methods gave almost identical results as far as the kinetics of virus D N A synthesis
are concerned. Virus D N A synthesis was found to attain its maximum in the middle of S
phase and virus and cellular D N A synthesis proceeded almost synchronously. Virus D N A
synthesis was only slightly delayed in cells infected up to Iz h after having been serumstimulated to progress to S phase, when compared to cells infected immediately after serum
stimulation. However, when cells were infected later than I3 h after serum stimulation,
when they had already entered S phase, a change to a delayed synthesis occurring in the
ensuing S phase was observed in all cases.
These results seem to show that the early virus information needed to initiate virus D N A
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I
r
I
~f
•
45 -
43I
I
1
#..........
,x
/~,r-i.--"""_..O
- 3
//if;i/i:ill
///
.~
>,30
m
-
×
-2
>
15
/
I
t
I
6
12
//I
1
\
t
~
18
24
30
Time of incubation (h)
1
-
~
-
-
-
36
Fig. 1. Whole cellular and virus D N A synthesis in GI arrested cells infected by polyoma virus and
incubated in different media. The method for determining cellular and virus D N A synthesis was described in Table i. [] . . . . [], Virus D N A yield in cultures incubated with fresh medium; © . . . . ©,
virus D N A yield in cultures incubated with medium pre-incubated with growing cells for 3 days;
A . . . . A, virus D N A yield in cultures incubated with ioo #M-5'-deoxy-5'-S-isobutyl adenosine/ml
serum free medium; m - - I I , rate of thymidine incorporation in whole cellular D N A in cultures
incubated with fresh medium; 0 - - 0 ,
rate of thymidine incorporation in whole cellular D N A
in cultures incubated with medium pre-incubated with growing cells for 3 days; J , - - J , , rate of
thymidine incorporation in whole cellular D N A in cultures incubated with zoo pM-5'-deoxY-5'S-isobutyl adenosine/ml serum free medium. Each point represents the mean values from three
Petri dishes.
replication must be expressed before a critical period of the S phase is attained
(Eckhart, ~974). From the shortening of the latency period observed in cells which were
infected 12 h after having been stimulated we can calculate that the expression of this early
virus function can occur in less than 9 h, which agrees with the findings that polyoma virus
penetrates into the nucleus in about ] 5 rain and that transcription of the early virus genome
is completed in about 6 h (Salomon et aL I977).
The similar yields and kinetics of virus DNA synthesis recorded in cells infected before
and IZ h after serum stimulation indicate that serum stimulation factors can efficiently
replace the virus host DNA stimulation function which is believed to be an early virus
function distinct from that of the TsA needed to initiate virus DNA replication (Eckhart,
1974). With regard to this, one has to consider that the majority of quiescent cells are probably in a similar state of GI and that, regardless of the type of stimulation, they have to
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432
Short communications
progress through a defined chemical programme, which cannot be shortened, before cellular
D N A synthesis can start (Pardee, I974). A striking parallel between the stimulatory effects
of serum and polyoma virus infection has already been found concerning shifts in enzyme
activities as well as in pool size of deoxyribonucleoside triphosphates (Nordenskjold et al.
I97o). We found no great changes in the kinetics of cellular D N A synthesis in either
stimulated infected or non-infected cells. However, the present cell system will be suitable
for the examination of possible qualitative changes, such as the order of mouse satellite
D N A synthesis (Smith, I97O; Balazs et al. 1973; Hatfield & Walker, I973). In a second
group of experiments, we tried to dissociate cellular and virus D N A synthesis. Serum stimulation of contact inhibited cells is due to as yet undefined serum proteins. We have found that
such factors can be eliminated from serum which has been in contact with growing cells for
different periods of time. When cells were incubated in pre-absorbed serum which had lost
the stimulation factors for cell replication, unimpaired yields of vesicular stomatitis virus
were obtained, indicating that the serum still contained essential nutritional factors.
Similar yields of polyoma virus D N A were also obtained in ceils incubated either with
fresh or pre-absorbed serum and in both cases almost all cells became productively infected
as judged by the capsid antigen staining method (Fig. I). This result is unexpected as fresh
serum stimulates all cells to replicate whereas this stimulation was almost completely absent
in cultures incubated with pre-absorbed serum.
This is a novel situation for polyoma virus and recalls what has been observed when
BSC-I cells were infected with SV4o (Ritzi & Levine, I97o). As results with pre-absorbed
serum sometimes varied, we recently combined this method with the use of either 5'-deoxy5'-S-isobutyl adenosine (Fig. I), an inhibitor of cell transformation (Robert-Gero et al.
I975), or methioninyl adenylate, a potent and reversible inhibitor of protein synthesis
(Cassio et al. 1973). However we can conclude from our observations that complete duplication of cellular D N A is not obligatory for virus D N A synthesis. Our cell synchronization
system will now be used to test the hypothesis that virus D N A synthesis may rely on biochemical programming characteristic for the S phase, which could be attained even in the
absence of significant replication of host DNA.
M . P . LOCHE
Fondation Curie-Institut du Radium
I5, rue Georges Cldmenceau
Bdtiment I IO
91405 - Orsay, France
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