J. gen. Virol. (I976), 30, I67-t77
I67
Printed in Great Britain
Induction of Cellular D N A Synthesis
and Increased Mitotic Activity in Syrian Hamster Embryo Cells
Abortively Infected with Human Cytomegalovirus
By T. A L B R E C H T , * M. N A C H T I G A L , ' ~ S. C. ST J E O R AND
F. RAPP~
The Department of Microbiology, The Milton S. Hershey Medical Center,
The Pennsylvania State University College of Medicine,
Hershey, Pennsylvania I7o33, U.S.A.
(Accepted 7 October I975)
SUMMARY
The effect of human cytomegalovirus (CMV) on cell DNA synthesis and mitotic
activity in hamster embryo fibroblasts was examined. The results indicated that
CMV infected cells had increased rates of cell DNA replication and mitotic
activity. Detection of the effect of CMV on these two parameters necessitated
arrest of cells prior to infection with low serum concentrations. This lowered the
background levels of DNA synthesis and cell division so that the effect of virus
infection could be detected. The data indicate that cells arrested prior to infection
demonstrate increased susceptibility to virus infection. It was also observed that
the effect of CMV on both DNA replication and mitotic activity could be enhanced
by irradiation with ultraviolet light of the virus prior to infection.
INTRODUCTION
It has been well established that cytomegaloviruses (CMV) abortively infect a number of
cell types (Kim & Carp, I97I ; Fioretti et al. I973; Waner& Weller, I974). In a recent study
involving transformation of hamster embiyo fibroblast (HEF) cells by human CMV, it was
observed that CMV is capable of both cell transformation and the induction of cytopathology in HEF ceils (Albrecht & Rapp, 1973). Other evidence obtained in this same investigation indicated that human CMV would not replicate in HEF cells. Considering the ability
of CMV to transform HEF cells, as well as the reported stimulatory effect of CMV on cell
DNA synthesis (St Jeor & Rapp, I973b, St Jeor et al. I974), we report the results of our
investigations on the apparent abortive infection of CMV in HEF cells.
METHODS
Cells. The methods used for preparation of HEF and human embryonic lung (HEL)
cells were, in general, those described previously (St Jeor & Rapp, I973b; Albrecht &
Rapp, I973). Eagle's medium supplemented with IO % foetal calf serum, o'o75 % sodium
* Present address: Department of Tropical Public Health, School of Public Health, Harvard University.
665 Huntington Avenue, Boston, Massachusetts o2~ I5.
~" Present address: University of Craiova, Craiova, Romania.
To whom reprint requests should be addressed.
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T. A L B R E C H T A N D O T H E R S
bicarbonate, Ioo units of penicillin and Ioo #g of streptomycin/ml was used for cell propagation. Secondary cultures of H E F and H E L cells between passages 5 to I5 were used in
these studies.
Arrest of cell growth. For experiments requiring cells with minimal D N A synthesis,
primary H E F were trypsinized and passed at a 1:3 ratio (based on surface area) to glass
prescription bottles or glass coverslips using Eagle's medium supplemented with o ' 5 %
foetal calf serum. Seventy-two to 96 h later these cells had formed about a 9o % confluent
monolayer with few, if any, mitotic figures. At this time the medium was poured off and
reserved, and the cells were either virus or sham inoculated. After I h adsorption the
inocula were removed and the cells washed twice with tris-buffered saline. The reserved
medium was then added back to the cell cultures.
H E F cells were arrested in the following manner to examine the effect of CMV on the
mitotic index: the cells were dissociated with trypsin and passed at a 1:3 ratio (based on
surface area) to 35 mm plastic tissue culture dishes containing I8 mm glass coverslips.
Eagle's medium supplemented with 5 % foetal calf serum was employed for these experiments. Forty-eight h after seeding, the cells had formed a confluent monolayer. The growth
medium was then removed and reserved. The cells were sham or virus inoculated and after
i h adsorption, the inocula were decanted. The cells were washed twice with tris-buffered
saline and a portion (I.5 ml) of the reserved growth medium was restored to each dish.
Virus and infectivity assay. The C-87 strain (Benyesh-Melnick, Rosenberg & Watson,
I964) of human CMV was used exclusively throughout these studies. The methods used for
virus propagation and assay have been published previously (Albrecht & Rapp, ~973).
Ultraviolet (u.v.) irradiation. Virus stocks were irradiated in I"5 ml portions with a GEG8T5 bulb at a dose rate of either 40 or 80 ergs/s/mm 2. The dose rate was determined with a
Blak-ray ultraviolet meter (UltraViolet Products, Inc., San Gabriel, California).
Fluorescent antibody assay. The indirect immunofluorescent (IF) assay was used to detect
CMV antigens. Human convalescent sera reactive with either CMV [reactive with varicellazoster (VZ), non-reactive with herpes simplex virus (HSV)] or with VZ (non-reactive with
either CMV or HSV) were used in these tests. Rabbit anti-human gamma globulin (H and L
chain specific) labelled with fluorescein isothiocyanate was purchased from Hyland, Costa
Mesa, California. The conditions employed for these assays have been reported (Albrecht
& Rapp, I973).
Virus growth kinetics. Confluent monolayers of cells were prepared in 25 cm 2 plastic
tissue culture flasks. After growth medium was removed, the monolayers were washed once
with tris-buffered saline. Virus (o'5 ml) diluted to obtain the stated multiplicity of infection
was adsorbed to the cells for I h at 37 °C with occasional rotation. The monolayers were
then washed three times with tris-buffered saline and maintenance medium (Eagle's supplemented with 2 % foetal calf serum) was added to each flask (5 ml/flask). All flasks were
incubated at 37 °C. At appropriate times, individual flasks were removed, quick frozen in
ethanol-dry ice, and stored at 76 °C. The cells were twice frozen and thawed, sonicated for
30 s with a Bronson model D-5o bath sonicator, and the debris pelleted by centrifuging at
2o0 g for Io min. The infectivity of the various samples was assayed as previously described.
Isotopic labelling and DNA analysis. The methods used for labelling and analysis of D N A
are described in earlier reports (Crouch & Rapp, I972 ). Isotope concentrations of IO #Ci/ml
of [~H]-methyl thymidine ([aH]-TdR, sp. act. t7 Ci/mmol) and 2o/~Ci/ml of [8-~H]-hypoxanthine (sp. act. I9"8 Ci/mmol) were used. All isotopes were obtained from SchwarzMann. Samples labelled with [3H]-hypoxanthine after D N A extraction were treated with
0. 3 M-KOH at 37 °C to hydrolyse labelled RNA.
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Abortive infection with human C M V
169
Fig. I. Photomicrograph o f non-arrested hamster embryo fibroblasts, 24 h p.i. with human cytomegalovirus (3 p.f.u./cell). Stained with haematoxylin and eosin.
Measurement of the mitotic index. The mitotic index of infected and control cultures was
determined using partially arrested cells (previously described). The cells were infected with
u.v.-irradiated or non-irradiated virus. Control cultures were inoculated with medium containing the same percentage of serum as the virus-infected cell lysate. After a I h adsorption,
the inoculum was removed and the monolayers were washed three times with tris-buffered
saline. The reserved culture fluids were then dispensed to each culture dish. At various
intervals post infection (p.i.), paired coverslips of infected and control cells were removed
and washed twice in tris-buffered saline. The cells were fixed with absolute methanol and
stained with z % Giemsa solution. Randomly chosen microscopic fields were examined with
oil immersion objective and the number of resting cells and mitotic figures was recorded. The
mitotic index was expressed as the ratio between cells in different stages of mitosis and ceils
in interphase.
RESULTS
Cytopathology in arrested and non-arrested HEF cells
Earlier studies reported from this laboratory indicated that when ceils were arrested by
either low serum concentrations or 5-iodo-z-deoxyuridine (IUdR) there was an increase in
cellular D N A synthesis when CMV infected cells were compared to control cells (St Jeor
et al. I974). A preliminary study was initiated to determine what effect the arrest of H E F
cells with low serum concentrations would have on the development of virus cytopathology
(c.p.e.). H E F cells, either arrested as described in the Methods section or non-arrested, were
infected with CMV at an input multiplicity of 3 p.f.u./cell. The cells were then examined
I2:
VIR 3°
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I70
T. ALBRECHT AND OTHERS
Fig. z. Photomicrograph of hamster embryo fibroblasts arrested by serum starvation, 24 h p.i. with
human cytomegalovirus (3 p.f.u./cell). Stained with haematoxylin and eosin.
for the development of both c.p.e, and CMV antigens. At 24 h p.i., c.p.e, was observed
in only about ~ % of the infected non-arrested cells. The c.p.e, consisted of a rather homogeneous cytoplasmic inclusion and an eccentric nucleus (Fig. ~). A few small rounded cells
were also observed at this time. The number of cells with c.p.e, increased only slightly 24 to
48 h after infection.
In contrast, the c.p.e, observed in infected, arrested cell cultures was extensive 24 h after
infection. Greater than 4o % of the cells were lost from the surface of the coverslips. This
was more pronounced than loss (~o to ~5 %) in arrested, non-infected cells. About 40 % of
those cells remaining had cytoplasmic inclusions and an eccentric nucleus (Fig. 2). Some of
the cells with inclusions were round, but most were fibroblastoid and many of the cells
(about 80 %) were refractile. By 48 h p.i., less than 1o % of the infected arrested cells remained attached to the glass and extensive c.p.e, was evident in nearly all of these cells.
Detection o f C M V antigens in arrested and non-arrested H E F
Preparations of infected and control arrested and non-arrested H E F cells were examined
for the presence of CMV antigens using the indirect IF test. When human convalescent
serum reactive with CMV was used in this test with infected non-arrested cells, a diffuse dim
cytoplasmic fluorescence was observed in about 1 % of the cells. However, when infected
arrested cells were examined, a very bright fluorescence was observed in about 9o % of the
cells (Fig. 3). The nucleus, cytoplasmic inclusions, and cytoplasm all fluoresced, although
not with equal intensity. Fluorescence was not observed with control arrested and non-
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Abortive infection with human C M V
17 t
Fig. 3. Fluorescence photomicrograph of hamster embryo fibroblasts arrested by serum starvation,
24 h p.i. with human cytomegalovirus (3 p.f.u./cell). These cells were fixed with cold acetone for
io min and stained with human convalescent serum reactive with cytomegalovirus and with
fluorescein isothiocyanate conjugated rabbit anti-human gamma globulin.
Table I. Assay for cytomegalovirus antigens in arrested and non-arrested
hamster embryo fibroblasts*
Condition
of cells
Virus
inoculated
~
Specificity of antisera?
~ -~
CMV~
VZ§
Arrested
+
+ (9° %)11
Non-arrested
+
+
(I
%)11
---
* Ceils were fixed in cold acetone for io min.
? Human convalescent.
~: Reactive with CMV and VZ but not HSV.
§ Reactive with VZ but not HSV or CMV.
rl Percentage of cells positive in the IF test.
arrested H E F cells a n d C M V reactive serum, n o r with infected a n d control, arrested or
n o n - a r r e s t e d H E F cells a n d V Z reactive serum (Table I).
Pre-treatment of H E F with xoo/~g/ml of I U d R (St Jeor & Rapp, 1973 a) yielded results
similar to those observed in serum arrested cells. However, the cytopathology was n o t as
extensive a n d the fluorescence with a n t i - C M V serum was n o t as bright as that observed i n
serum arrested cells.
Adsorption and rate of inactivation of C M V in arrested and non-arrested cells
A possible explanation for the difference observed between arrested a n d n o n - a r r e s t e d
cells infected with C M V might be that the virus either adsorbed more rapidly to the arrested
12-:2
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4:
T. ALBRECHT AND OTHERS
172
I
I
I
I
l
l
l
l
l
l
3(
E
~2
1
ol
o 10 20 30 40 50 60 70 80 90 100
Time post infection (h)
Fig. 4. Kinetics of human cytomegalovirus inactivation in arrested ( 0 - - 0 ) and non-arrested
(©--©) hamster embryo fibroblasts. Monolayer cell cultures were inoculated at an input multiplicity of 2 p.f.u./cell and were harvested at the times indicated by two cyclesof rapid freezing and
thawing followedby sonication. The infectivity of each sample was determined by plaque formation
in monolayers of human embryonic lung cells.
cells or was inactivated more rapidly in the non-arrested cells. To test this hypothesis the
following study was initiated. Cultures of arrested and non-arrested H E F cells were prepared in I oz glass prescription bottles and inoculated with CMV at an input multiplicity
of 2 p.f.u./cell. Following a 1 h adsorption period, the infected cells were washed 3 times with
tris-buffered saline. The amount of virus infectivity present in the washes from the cells was
determined by a plaque assay to determine differences in the efficiency of virus adsorption
in arrested and non-arrested ceils. The results of this study indicated that > 9o % of the
virus in both the arrested and non-arrested cells remained adsorbed to the cells throughout
the washings. The rate of inactivation of the virus in arrested and non-arrested cells was then
determined by assaying cultures for infectious virus at various periods following infection.
The inoculum decay curves (Fig. 4) in arrested and non-arrested cells were similar for
about o to 48 h following infection. Thereafter, the infectivity was lost more rapidly from
the arrested cell cultures than from the non-arrested cell cultures. At no time was an increase
in virus infectivity above input levels observed.
Effect of C M V on H E F cell DNA synthesis
Earlier reports from this laboratory indicated that CMV induced increased cell D N A
synthesis in both productive and abortive infections in a variety of human cell types (St
Jeor & Rapp, I973a, b; St Jeor et al. 1974). In order to determine the effect of CMV on
D N A synthesis in H E F cells, the following study was initiated. H E F cells, either arrested
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Abortive infection with human C M V
20
18
16
7 14
×
12
e,
10
"d 8
6
4
2
0
I73
x1!I
I
24
i
I
i
I
48
72
96
120
Time post infection (hi
Fig. 5
0-24 h p.i.
24 48 h p.i.
Time post infection
Fig. 6
Fig. 5. Kinetics of cellular DNA synthesis in non-arrested hamster embryo fibroblasts. Cell cultures
were inoculated with non-irradiated human cytomegalovirus (O--D), ultraviolet irradiated
(24ooo ergs/mm~) cytomegalovirus (&--&), or with maintenance medium (Z] []) containing the
same concentration of foetal calf serum as the virus inoculum. The cell cultures were pulse-labelled
with [3H]-TdR as described. Each datum point represents the total radioactivity incorporated into
cellular DNA during a 24 h interval and is indicated at the midpoint of the pulse period. DNA was
analysed as described in the text.
Fig. 6. Kinetics of cellular DNA synthesis in arrested hamster embryo fibroblasts. Total radioactivity
incorporated into cellular DNA in cells infected with non-irradiated cytomegalovirus (hatched
bars), ultraviolet irradiated cytomegalovirus (solid bars), or control cultures (open bars) inoculated
with maintenance medium containing the same concentration of foetal calf serum as the virus
inoculum was measured. The conditions employed in analysing the DNA are identical to those
described in Fig. 5.
(as described ~previously) or non-arrested, were infected with u.v. irradiated or nonirradiated C M V or sham infected with medium. The cultures were labelled for consecutive
24 h periods with [3H]-TdR. Following the labelling periods, D N A was extracted as described and ~centrifuged to equilibrium in CsCI gradients. A single peak o f radioactive
labelled molecules was observed at the density o f H E F cell D N A (I.699 g/ml). There were
no labelled molecules detected with a density corresponding to C M V D N A (I.716 g/ml).
Significant differences in the rate o f cell D N A synthesis between non-arrested infected and
uninfected cells were not detected (Fig. 5); however, differences were observed in the uptake
o f [3HI-methyl thymidine into cellular D N A in arrested cells (Fig. 6). F r o m o to 24 h p.i.,
a b o u t three times as m a n y counts were incorporated into cellular D N A in infected cell
cultures than in control cell cultures. F r o m 2A to 48 h p.i., the level of isotope incorporated
in virus infected cell cultures fell to the level o f control cultures; however, the incorporation
o f labelled thymidine in irradiated virus infected cell cultures was about 4"5 times greater
than the level in control cultures.
Since there is the possibility that C M V infection m a y alter the relative p r o p o r t i o n o f
endogenously synthesized and exogenous thymidine available for D N A synthesis, these
experiments were extended using [3H]-hypoxanthine and serum-arrested cells. The results o f
this experiment for the 24 to 48 h p.i. period are summarized in Fig. 7. As in the previous
experiments, the level o f D N A synthesis in C M V infected cultures at 24 to 48 h was less
than that observed in control cultures. However, cell cultures infected with virus irradiated
with 24ooo ergs/mm ~ o f u.v. light had about a threefold greater rate o f D N A synthesis
during this period than that observed in control cultures. Cell cultures inoculated with
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T. A L B R E C H T
174
4.0
o
l
I
I
AND OTHERS
I
I
I
3-0
3
2.0
E
1.0
I
I
I
I
I
I
2"4
4"8
7"2
9"6
12"0
14"4
u.v. dose, ergs/mm2×10 4
Fig. 7. Kinetics of cellular D N A synthesis in arrested hamster embryo fibroblasts as a function of
ultraviolet irradiation dose. laH]-hypoxanthine was used to measure the rate of cellular D N A
synthesis during 24 h intervals from o to 48 h p.i. The D N A was released by the sarkosyl-EDTApronase procedure used previously (Crouch & Rapp, I972). The total alkali-stable, acid-precipitable
counts were determined and are indicated here as fraction of the control for the 24 to 48 h p.i.
period only.
virus exposed to greater doses of irradiation demonstrated decreased levels of isotope
incorporated into cellular DNA when compared with cells inoculated with virus irradiated
with 24ooo ergs/mm ~. With doses equal to or greater than 96 ooo ergs/mm z, the stimulating
activity of the virus inoculum was lost.
Mitotic activity of HEF following infection with C M V
Stimulation of cell DNA synthesis by CMV represents new semi-conservative DNA
synthesis (St Jeor et al. 2974). In order to determine whether the increased DNA synthetic
activity would be succeeded by increased mitotic activity, the following study was initiated.
The methodology employed in reducing the background of cellular DNA synthesis to levels
where stimulation of cellular DNA synthesis might be detected could prohibit cells from
subsequently entering mitosis. Therefore, a protocol (previously described) was used which
allowed cells to enter mitosis following viius infection. Employing these cell cultures and
virus stock treated with various doses of u.v. light, increased mitotic activity was demonstrated following CMV infection (Fig. 8). The greatest stimulation of mitotic activity (up to
sixfold) in cell cultures infected with u.v.-irradiated stock (24ooo ergs/mm ~) was observed
in the initial 24 h p.i. period. Other doses of irradiation of the virus inoculum gave lower
stimulatory effects. By 48 h p.i., the level of mitotic activity in cell cultures inoculated with
irradiated virus was at the level of the control. In contrast, little increase in mitotic activity
was observed in cell cultures inoculated with non-irradiated virus.
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Abortive infection with human C M V
I75
60 I---
25-
I
I
V2.5'u.v.
50
I_
40
20
.CMV 10' u.v.
o
30
15
r,
CMV 30' u.v.
-~ 10
20
5
10
Control
/
•
~'-CMV 0' u.v.
\
I
16
I
24
Time post infection (h)
48
0 •
0
16
24
Time post infection (h)
Fig. 8
Fig. 9
Fig. 8. Mitotic index of hamster embryo fibroblasts inoculated with non-irradiated human cytomegalovirus ( 0 - - 0 ) ; with ultraviolet-light-irradiated cytomegalovirus: i2ooo ergs/mm ~ (A--A),
240o0 ergs/mm 2 (Ak--A), 48ooo ergs/mm 2 ([]--[]), ~44ooo ergs/mm 2 (11--11).; or with maintenance medium ( ( 3 - - 0 ) containing the same concentration of foetal calf serum as the virus
inoculum. The cells were fixed with absolute methanol and stained with Giemsa. Cells were counted
fi'om 4oo microscopic fields to determine each datum point.
Fig. 9. Mitotic index of hamster embryo fibroblasts in the presence of colcemide (r #g/ml). Cell
cultures were inoculated with non-irradiated human cytomegatovirus ( 0 - - 0 ) ; with ultravioletlight-irradiated cytomegalovirus: 12o0o ergs/mm 2 (A--A), 48o0o ergs/mm 2 (Q--[]), 14400o ergs/
mm ~ (11--11); or with maintenance medium (Q--O) containing the same concentration of foetal
calf serum as the virus inoculum. The cells were fixed with absolute methanol and stained with
Giemsa. Cells were counted from 2oo microscopic fields to determine each datum point.
The increase in mitotic figures observed at 48 h p.i. in the cultures inoculated with n o n irradiated C M V could represent a release of contact i n h i b i t i o n for cellular division by
massive destruction in the confluent m o n o l a y e r created by virus-induced cell death with the
s u b s e q u e n t arrest of cells in mitosis following virus infection. Therefore, the experiments
were repeated i n the presence of colcemide (~ #g/ml). The results of this experiment are
graphed in Fig. 9- S t i m u l a t i o n of mitotic activity was observed in all cell cultures i n o c u l a t e d
with irradiated virus. However, the greatest mitotic activity was observed i n cultures
inoculated with virus irradiated with 12ooo ergs/mm ~ of u.v. light. Increasing doses of u.v.
i r r a d i a t i o n progressively diminished the stimulating effect u p o n mitotic activity. The
mitotic index of cultures inoculated with n o n - i r r a d i a t e d virus never exceeded that of the
controls.
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I76
T. A L B R E C H T
AND
OTHERS
DISCUSSION
Qualitatively, the cytopathology of CMV infected HEF is very similar to that reported
by Waner & Weller (I974) in bovine cells and by Fioretti et aL 0973) in guinea-pig cells.
The cytopathology observed in these cell types (all of which are abortively infected) appears
early (5 to 24 h p.i.), and consists of refractile cells, cytoplasmic inclusions and eccentric
nuclei. Nuclear inclusions, as observed in productively infected HEL, and prominent multinucleated giant cells, as observed in CMV inoculated Vero cells (Waner & Weller, I974),
were absent from CMV inoculated HEF.
Quantitatively, however, CMV infection of HEF cells appears to be different from the
other cell types (bovine, guinea pig) sustaining abortive infections. Waner& Weller (I974)
found cytopathology in about 4o ~o of CMV infected bovine cells and CMV antigens in a
similar percentage of inoculated cells. Similar results were obtained by Fioretti et al. 0973)
in guinea-pig cells. In contrast, cytopathology and virus antigens were observed in only
about 1% of CMV infected non-arrested hamster cells. Furthermore, while both Fioretti
et aL 0973) and Waner& Weller (t974) found nuclear and cytoplasmic antigens in their
respective cell types, only cytoplasmic antigens were detected in infected non-arrested
hamster cells. However, if the hamster cells were arrested by serum starvation, then cytopathology was observed in the vast majority of inoculated cells and virus antigens were
present in the nucleus and cytoplasm.
Cellular DNA synthesis was also affected by CMV infection. The degree of stimulation
observed was about 5o % of that previously reported in other cell types (HEL, Vero) by St
Jeor et al. 0974) which sustain productive and inefficient infections, respectively (Waner &
Weller, 1974).
Since stimulation of cell DNA synthesis was observed in these cells, it is not surprising to
find an increased level of mitotic activity. Lang, Montagnier & Latarjet (I974) also observed
increased mitotic activity following CMV infection. The observed increases in the mitotic
index do not appear to be the result of cells arrested in mitosis, since there is an accumulation
of mitotic cells in the presence of colcemide. It is interesting to note that at least a fivefold
greater dose of u.v. light to the virus stock is required to eliminate the induction of DNA
synthesis and mitosis, than to eliminate cytopathology. Apparently, the test for mitosis (on
individual cells) is somewhat more sensitive than stimulation of DNA (on cell populations)
as a measure of CMV activity.
These data indicate that CMV is capable of inducing both DNA synthesis and mitosis in
hamster embryo cells. Earlier studies (St Jeor & Rapp, I973a, b; St Jeor et aI. I974) indicated
that CMV can induce cell DNA synthesis in a variety of cell types. Studies in this laboratory
(S. C. St Jeor & J. J. M. Walboomers, unpublished experiments) indicate that CMV, rather
than coding for a virus distinct thymidine kinase, induces higher levels of cellular thymidine
kinase. It is therefore possible that the replication of CMV is dependent upon the cell
cycle.
We thank Dr Fred Funk for his many helpful suggestions and criticism and Dr John
Kreider for his evaluation of various cell preparations and very useful suggestions and
criticism. This work was supported by Contract No. NOI CP535~6 within The Virus Cancer
Program of the National Cancer Institute and by a predoctoral fellowship to Thomas
Albrecht.
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Abortive infection with human C M V
I77
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