6i
J. gen. Virol. 0972), I6, 61-68
Printed in Great Britain
Virus Infection as a F u n c t i o n o f the H o s t Cell Life Cycle:
Replication o f Poliovirus R N A
By T A M I L L A E R E M E N K O , A. B E N E D E T T O AND P. V O L P E
International Institute of Genetics and Biophysics, Via Marconi I2, Naples, and
Centre for Virology, OORR, Rome, Italy
(Accepted I5 March I972)
SUMMARY
The rates of synthesis and final yields of poliovirus RNA varied considerably
during the four main phases (G1, S, Gz and m) of the life cycle of synchronized
HeLa cells. The rate of RNA synthesis late in virus growth (as measured by uridine
incorporation 2 to 4 hr after infection) and the final yield of RNA rose sharply if
growth was initiated towards the end of phase S; that is, RNA was synthesized most
rapidly if cells were infected during the period of most rapid DNA synthesis. In
contrast, the initial rate of RNA synthesis (incorporation 0 to 2 hr after infection)
was greatest if growth was initiated at the end of phase G~, just before mitosis.
This differential effect on growth kinetics suggests that the balance between the two
stages of virus RNA synthesis (production of complementary minus strands and of
progeny plus strands) is dependent on unknown cellular factors.
INTRODUCTION
The relationships between host cell and infecting poliovirus have been a major area of
research for the last ten years. The influence of the infectious process on macromolecular
synthesis in the host is relatively well known (Penman & Summers, 1965; Darnell et al.
1967). The relation between the cellular metabolic state and the mechanism of virus replication has been less studied.
One of the most promising approaches to this problem is the use of synchronized HeLa
cell cultures allowing accurate monitoring of each phase of the mitotic cycle (Volpe &
Eremenko, 197oa) which corresponds to well defined metabolic states (Robbins & Scharff,
I965; Volpe & Eremenko, I97ob, ~971). We have used poliovirus as infecting agent in HeLa
cells (Eremenko, Benedetto & Volpe, I970, since this system has been characterized from
the point of view of genetics (Cooper, 1969), RNA metabolism (Darnell et al. 1967; Jacobson
& Baltimore, 1968; Noble & Levintow, 197o), coat protein structure and antigenicity
(Katagiri, Hinuma & Ishida, 1968) and inhibiting factors (Caliguiri & Tamm, x968; Pearson
& Zimmerman, 1969; Cooper, Wentworth & McCahon, I97O; Hecht & Summers, 197o). In
this paper we describe the different patterns of replication of virus RNA in the various
phases of the life cycle of HeLa cells.
5
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62
T. E R E M E N K O , A. B E N E D E T T O
A N D P. V O L P E
METHODS
Cell cultures and media. Suspension cultures of HeLa $3 cells were subcultured on alternate
days using Minimum Essential Medium (MEM) as modified by Joklik (GIBCO Catalogue
No. I2-616), without Ca 2+, and containing Io % calf serum (Volpe & Eremenko, I97ob). Cell
density was kept between o.25 and o'5 x Io Gcells/ml. The suspensions were maintained in
spinners (Bellco Glass) at 37 ° + 0.2 and aerated by a constant flow of 5 % CO2 in air.
Cell synchronization. Cells were synchronized according to Puck (2964). Cells were sedimented in the Sorvall GSA rotor at 2ooo rev./min, for 2o min. and resuspended in I 1. of
medium containing 2 m-mole-thymidine to initiate the synchronization at a density of
o'5 × IO6 cells/ml. After 24 hr the cells were sedimented, suspended in fresh medium without
thymidine and left to grow under these conditions for 8 hr. At this time a second thymidine
shock was given after which the cells, placed into normal medium, entered the S-phase. The
degree of synchronization achieved was indicated by the density of the culture which
remained at o'5 × 208 cells/ml. As soon as the cells were replaced in the medium without
thymidine, the length of each phase of the cellular cycle was measured according to the
method described earlier (Volpe & Eremenko, ~97oa).
Conditions of infection and labelling of virus RNA. A cloned strain of poliovirus was
grown on HeLa S3 cells, purified in a CsC1 gradient with minor modifications of the Levintow
& DarneU method (I96O) and assayed on monolayers of 37 RC cells (Djaczenko, Benedetto
& Pezzi, 297o).
With asynchronous cultures, as well as synchronous cultures at the different stages of the
cell cycle, I5 x 206 cells were collected, sedimented in the Sorvall SS-34 rotor at 2ooo rev./min.
for 5 rain. and resuspended in 2o ml. of MEM containing 2 % calf serum, the purified virus,
Io #g./ml. actinomycin D ando'5 × 2o-2 M-guanidine. The cells were infected at 8o p.f.u./cell.
The poliovirus-HeLa cell mixture was incubated for I hr at 37 ° with stirring and kept
aerated with 5 % CO2. At the end of the incubation the cells were washed twice in cold MEM
and resuspended in 2o ml. of preheated medium containing 2 #c/ml. [3H]-uridine. Starting
from the first hr, samples of I ml. were collected every 3° min. up to 5 to 8 hr and analysed
for radioactive R N A according to the following method. The suspension was centrifuged for
5 min. at 2ooo rev./min, in the Sorvall SS-34 rotor after addition of I ml. of cold MEM. The
harvested ceils were resuspended in 2 ml. of distilled water and dissolved in I ml. of a
solution of SDS-EDTA (o.o2 M-NaC1; o.oi M-tris HC1 p H 7; 1% sodium dodecyl sulphate
and o'o5 M-EDTA). Suitable samples were brought to 7 Yo with TCA, left in ice for 2o min.
and filtered on millipore HA o'45/zm, with 5 % TCA. The determination of the radioactivity was made in Bray's solvent using a Nuclear Chicago scintillation spectrometer.
Virus and chemicals. Type 2 (Mahoney) non-purified poliovirus was supplied by Sclavo
Italiana. The Joklik modified MEM for spinners and calf serum were furnished by GIBCO.
Before use, the serum was heated for 2 hr at 5o° and filtered on Seitz EKS II. Unlabelled
thymidine, actinomycin D and guanidine were obtained from SIGMA; [ZH]-thymidine
(6"7 c/m-mole) and tritiated uridine (25 c/m-mole) were supplied by New England Nuclear.
CsC1 was from Merck.
RESULTS
Cell synchrony
The length of each phase of the mitotic cycle was accurately determined in every experiment (Fig. I). Synchronization was achieved usually with I 1. suspensions of HeLa cells kept
at the density of o'5 × Io6/ml. Starting from the time of reversion to the medium without
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Poliovirus infection during the mitotic cycle
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Fig. I. Determination of the length of the four main phases of the HeLa cell life cycle. From the time
of entering the S-phase, at given intervals D N A synthesis (A) was followed treating the cells with
2 ,~c/ml. of [3H]-methyl thymidine (6"7 c/m-mole) for 20 min, at 37 °. The arrow in the cell cycle
schema indicates the time of replacement of synchronizing thymidine (see Methods).
Fig. 2. Replication of poliovirus R N A in asynchronous cultures of HeLa cells grown and infected
as described in Methods. Virus infection was induced at 80 p.f.u./cell. Virus R N A was labelled with
5 #c/ml. [SHl-uridine 25 c/m-mole (50//,clio m1./15 x IO6 cells).
thymidine, samples were taken at short intervals to measure DNA synthesis (Fig. I A), population density (Fig. I B) and rate of mitotic divisions (Fig. I C). The full length of the mitotic
cycle was about I9 hr, while the phases S, G2, m and G1 lasted 6, 4, I'5 and 7"5 hr,
respectively.
Virus RNA labelling in asynchronous cells
Fig. 2 shows the kinetics of poliovirus RNA replication in an asynchronous culture of
HeLa cells. The shape of the curve is very similar to that described in other laboratories
(Darnell et al. I967). During the first 4 hr, the synthesis of actinomycin D-resistant virus
RNA is linear; maximum labelling takes place at the 4th hr and is followed by a limited
linear decrease. Under our experimental conditions the value of maximum labelling of
poliovirus R N A in asynchronous HeLa cells is about 8oo counts/min./Io 6 cells.
5-2
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64
T. E R E M E N K O ,
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Fig. 3. Replication o f poliovirus R N A during the life cycle of I t e L a cells. Cells were grown and
synchronized as described in Methods. The length o f each phase o f the cell cycle was measured as
shown in Fig. I. Cell samples were infected at hourly intervals during the cell cycle with 80 p.f.u./cell
and virus R N A synthesis was followed for 4} hr under the same conditions o f Fig. 2.
Virus infection and virus RNA labelling during the cell cycle
Samples of synchronized HeLa cells were infected by poliovirus at hourly intervals during
the whole cell life cycle, and with each sample the kinetics of virus RNA labelling was
followed for 4} hr (Fig. 3). The multiplicity of infection and the labelling conditions were
kept the same in all samples.
Although in these experiments the cell cycle started after removal of the thymidine from
the culture medium, namely with phase S, the results are presented in the classical sequence
of the mitotic cycle, namely G1, S, G~ and m.
In the first hr of life of the Gl-cell (G0-phase according to Epifanova & Terskikh, ~969),
poliovirus RNA labelling developed linearly during 4} hr, reaching about I6oo counts•
min./w 8 cells. This value is twice that reached in asynchronous cultures (Fig. 2). From the
2nd to the 4th hr of the Grphase (early Gl-phase) the labelling of poliovirus RNA was no
longer linear and appeared to follow a bimodal trend. In the late G~-phase (from the 5th hr
onward) the maximum level of labelling gradually decreases to about 6oo counts/min./I@
cells, and the curve lost its bimodal shape, becoming asymptotic. During the Gl-phase, the
maximal level of labelling of virus RNA gradually decreased, while the shape of the kinetic
curves also changed.
When the cell entered the S-phase there was a gradual increase in the synthesis of
actinomycin D-resistant virus RNA which reaches the highest level in the cycle (about
3000 counts[min./w6 cells). The shape of the replication curve is somewhat similar to that
observed in asynchronous ceils. In this case, however, the replicative curve shows an initial
lag period of about z hr and reaches a maximum velocity after approximately 3 hr.
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65
Poliovirus infection during the mitotic cycle
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Fig. 4. Replication of poliovirus R N A during the H e L a cell karyokinetic p h a s e m. Cell samples
were infected every 3o min., a n d virus R N A synthesis followed for 8 h r thereafter. Experimental conditions were as for Fig. z a n d 3. T h e h o u r s s h o w n above the graphs refer to the times f r o m the
beginning of the Gl-phase.
Apparently the physiological state of the cell duplicating its own D N A strongly influences
the initiation of poliovirus infection.
As soon as the cell enters the Gz-phase, the kinetics of the poliovirus RNA labelling were
again markedly changed. The curve became slightly sigmoidal, and its level fell to about
8o0 counts/min./I@ cells. The shape of the curves was also quite similar to that established
in the early Gl-phase. The early Gl-phase and the late G2-phase would appear to provide
analogous conditions for the initiation of poliovirus infection. Moreover, during the
G2-phase, the initial velocity of virus RNA replication reaches the highest level in the cycle.
Phase m is characterized by a clear bimodal shape of the replicative curve of poliovirus
RNA and by a low level of labelling (Fig. 3 and 4). When the virus infections are made at
3° rain. intervals during the m-phase (Fig. 4), it can be noted that, while the first peak of the
bimodal curve developing in about 2 hr remained approximately constant, the second was
almost completely abolished at later times. Incidentally, at the beginning of the m-phase
(prophase) the level of labelling observed between the two peaks was the lowest of the cell
cycle (less than IOO counts/min./Io 6 cells). This decrease may correspond to the autoradiographic observation by Salb & Marcus (I965) in cells arrested in mitosis with vinblastine
sulphate.
While poliovirus RNA labelling started in one phase of the cell cycle and appeared to
continue into the adjacent one (Fig. 3), this might be merely an apparent feature stemming
from the necessity to present the data in sequence throughout the cell cycle. Virus infection
into a cell rapidly blocks macromolecular synthesis (Penman & Summers, 1965) and the
actinomycin D inhibits the synthesis of cell messenger RNAs. It may be presumed, thus,
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66
T. E R E M E N K O ~ A. B E N E D E T T O A N D P. V O L P E
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Fig. 5. Extent of poliovirus R N A labelling during the HeLa cell cycle, Each point was calculated using
the data of Fig, 3 and 4. The upper curve refers to the label incorporated into virus R N A during
the first two hr of incubation with radioactive uridine; the lower curve refers to the incorporation
occurring between the 2nd and the 4th hr. The abscissa shows the time at which each infection was
initiated.
that the infected cells do not proceed normally into the following phases but might be
arrested at the phase in which they were infected.
Rate of virus RNA synthesis during the cell cycle
Bimodal curves of poliovirus R N A replication occur in phases G1, G2, and, most
markedly, in the karyokinetic phase m. In all cases the two peaks in these curves appear at
similar times from the start of the incorporation. While the first peak develops in about 2 hr,
the second peak reaches its maximum after approximately 4 hr (Fig. 3 and 4). To elucidate
this we have examined throughout the whole cell cycle the amount of radioactive virus R N A
present after 2 hr and synthesized between 2 and 4 hr from the beginning of labelling
(Fig. 5). Virus R N A labelling in the first two hr decreased slowly if growth was initiated
during the G1- and S-phases and increased at the end of the G~-phase. The extent of incorporation into virus R N A in the period 2 to 4 hr varied much more reaching a sharp
maximum if growth was initiated towards the end of the period of cell D N A synthesis
(S-phase). It appears that particularly the late stages of virus R N A synthesis are strongly
dependent on the phases of the cell cycle.
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Poliovirus infection during the mitotic cycle
67
DISCUSSION
The experimental data presented in this paper show that the metabolic state of the cell,
identified by the phases of its life cycle, influences the kinetics of poliovirus RNA replication.
Different kinetics of poliovirus RNA replication are observed, in fact, in the G1-, S-, G2- and
m-phases and in different segments of the G1, G2- and m-phases as well. At the different
stages of the cell life cycle there are changes not only in the extent of maximal virus RNA
labelling which reaches its highest value in the S-phase and its lowest in the m-phase, but
also in the shape of the RNA replicative curve. Two main types of curves are present during
the cell cycle. The unimodal type reached a maximum after approximately 4 hr. The bimodal
curves had their first peak at about 2 hr and a second rise at about 4 hr.
At the moment, there is insufficient evidence to draw conclusions about the differential
synthesis of single-stranded, replicative intermediate and replicative forms of RNA (Noble
& Levintow, I97O). However, in considering the amount of radioactive precursor incorporated into virus RNA in the intervals o to 2 hr and 2 to 4 hr after infection, it is clear that the
second value is much more dependent on the phase of the cell cycle than the first one. This
might suggest that the production of the complementary strand of virus RNA is only partly
dependent on the phases of the cellular cycle, while the production of the progeny plus
strand is a strongly dependent variable bound to the phase of the cellular DNA synthesis.
Some data support the idea that the S-phase is particularly involved in virus infection
(Tennant & Hand, 197o; Hampton, I97O; Keserovic, I97I ; Lawrence, I971), while in other
cases the cell cycle does not influence the virus infection at all (Cairns, 196o; Groyon &
Kniazeff, I967; Hodge & Scharff, 1969). These two groups of data might reflect the different
dependency on the mitotic cycle observed in the present investigation for the early and late
stages of the virus replication. Alternatively, the dependency of the virus infection on the
cell cycle might vary with the type of the cell-virus system studied.
The authors wish to thank Prof. A. Giuditta, of the International Institute of Genetics and
Biophysics, and Dr E. Whitehead, of the Euratom Organization, for critical reading of the
manuscript. Thanks are also due to Mr C. Buono for skilful technical assistance, and to
Mr G. De Simone for laboratory work.
REFERENCES
CAIRNS, J. (I960). T h e initiation o f vaccinia infection. Virology IX, 603.
CALIGUIRI, L. A. & TAMM,I. 0968). Action of guanidine on the replication of poliovirus R N A . Virology35, 4o8.
COOPER, P. D. (I969). T h e genetic analysis of poliovirus. In The Biochemistry of Viruses. Ed. H i l t o n B. Levy,
P. I77.
COOPER, P. D., WENTWORTH, B. B. & McCAHON, D. (I970). G u a n i d i n e inhibition o f poliovirus : a dependence o f
viral R N A synthesis o n the configuration o f structural protein. Virology 4 o, 486.
DARNELL, J. E., GIRARD, M., BALTIMORE,D., SUMMERS,D. F. & MAIZEL, J. V. 0967). T h e synthesis a n d translation
o f poliovirus R N A . I n The Molecular Biology of Viruses, pp. 375-4oi. N e w Y o r k : A c a d e m i c Press.
DJACZENKO, W., BENEDETTO, A. & PEZZI, R. 097O)- F o r m a t i o n of helical polyribosomes in poliovirus-infected
cells o f the 37 R C line. The Journal of Cell Biology 45, ~73.
EPIFANOVA, O. I. & TERSKIKH, V. V. (I969). O n the resting periods in the cell life cycle. Cell Tissue Kinetics
2, 75.
EREMENKO, W., BENEDETTO, A. & VOLPE, P. (I97I). Initiation o f poliovirus replication. Second International
Congress for Virology (Budapest), Proceedings in press.
GROYON, R. M. & KNIAZEFE, A. S. (1967). Vaccinia virus infection o f synchronized pig kidney cells. Journal of
Virology x, I255.
HAMPTON, E. (I970). H - I virus growth in synchronized fat e m b r y o cells. Canadian Journal of Microbiology
x6, 266.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 14:53:22
68
T. E R E M E N K O , A. B E N E D E T T O A N D P. V O L P E
rlECHT, T. T. & SUMMERS,D. r. (I970). The effect of phleomycin on poliovirus R N A replication. Virology 4o,
44L
r~OD~E, L. D. & SCHARrF,M. D. (1969). Effects of adenovirus on host cell D N A synthesis in synchronized cells.
Virology 37, 554.
JACOaSON, M. r. & BALTIMORE,D. (I968). Morphogenesis of poliovirus : I. Association of the viral R N A with
coat protein. Journal of Molecular Biology 33, 369.
KATAGIRI,S., mIqUMA,V. & ISHIDA,N. (I968). Relation between the adsorption to cells and antigenic properties
in poliovirus particles. Virology 34, 797.
KESEROVlC,a. (I970. Dejstvo herpes simplex virusa na deobu L celija. International Symposium on Experimental Oncology (3rd Yugoslavian Congress of Cancerology), Zagreb, Abstracts, IV, 439.
LAWRENCE, W. C. 097I). Evidence for a relationship between equine abortion (herpes) virus deoxyribonucleic acid synthesis and the S phase of the KB cell mitotic cycle. Journal of Virology 7, 736.
LEVINTOW, L. & DARNELL,J. E. (I960). A simplified procedure for purification of large amounts of poliovirus:
characterization and amino acid analysis of type I poliovirus. Journal of Biological Chemistry 235, 70.
NOBLE, J. & LEVINTOW,L. (I970). Dynamics of poliovirus-specific R N A synthesis and the effects of inhibitors
of virus replication. Virology 40, 634.
PEARSON, G. D. & ZIMMERMAN, E. r . (1969). Inhibition of poliovirus replication by N-methylisatin-fl-4':4'dibutylthiosemicarbazone. Virology 38, 64I.
PENMAN, S. & SUMMERS,D. F. (I965). Effects on host cell metabolism following synchronous infection with
poliovirus. Virology 27, 614.
PUCK, T. "r. (I964). Studies on the life cycle of mammalian cells. ColdSpring Harbor Symposium on Quantitative Biology 29, 167.
ROBBINS, E. & SCrlARrF, M. (I965). Macromolecular synthesis in metaphase arrested cells. Federation
Proceedings 24, 445.
SALB, J. M. & MARCUS,P. I. (I965). Translational inhibition in mitotic HeLa cells. Proceedings of the National
Academy of Sciences of the United States of America 54, 1353TENNANT, R. W. & HAND, a. E. (1970). Requirement of cellular synthesis for Kilham rat virus replication.
Virology 42, IO54.
VOLPE,P. & EREMENI,:O,T. 0 970 a). A method for measuring the length of each phase of the cell cycle in spinner
cultures. Experimental Cell Research 60, 456.
VOLPE, P. & EREMENKO,T. 097ob). Quantitative studies on cell proteins in suspension cultures. European
Journal of Biochemistry I2, I95.
VOLPE, P. & EREMENI(O,T. 097I). Structural enzyme heterogeneity as expressed by differentiated regulation
during the phases of the cell cycle. In Advances in Cytopharmacology VoL z, p. 257. New York: Raven
Press.
(Received z I December I 9 7 I )
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