Download Fate of Frog Virus 3 DNA Replicated in the Nucleus of Arginine

J. gen. Virol. (1984), 65, 721-732. Printedin Great Britain
721
Key words : FV 3/arginine/DNA replication~nuclearlocalization
Fate of Frog Virus 3 DNA Replicated in the Nucleus of Arginine-deprived
CHO Cells
By J. P. M A R T I N , *
A. M. A U B E R T I N ,
L. T O N D R E
AND A. K I R N
Groupe de Recherches de r l N S E R M (U 74) sur la Pathogbnie des Infections Virales et
Laboratoire de Virologie de la Facultk de MOdecine, Universitb Louis Pasteur, 3 rue Koeberlb,
67000 Strasbourg, France
(Accepted 15 December 1983)
SUMMARY
In a productive infection, frog virus 3 (FV 3) D N A was synthesized in both the cell
nucleus and cytoplasm. The infection was aborted in arginine-starved Chinese hamster
ovary cells and viral DN A replication was then restricted to the nuclear compartment.
It has been demonstrated that the newly synthesized FV 3 D N A present in the nucleus
is of genomic size. After the addition of arginine, this D N A is transferred into the
cytoplasm and can be encapsidated. The formation of infectious particles occurred
even if an inhibitor of D N A synthesis was added simultaneously with arginine.
Although the synthesis of early FV 3 polypeptides and their intracellular distribution
were comparable in the presence or in the absence of arginine, the production of late
species was greatly reduced by arginine deprivation; one of these late proteins must be
involved in the passage from the nuclear to the cytoplasmic phase of FV 3 D N A
replication. This system made it possible to carry out an independent analysis of the
nuclear events that comprise the first stage of FV 3 multiplication.
INTRODUCTION
A novel pattern of frog virus 3 (FV 3) replication has recently been proposed. D N A replicates
in two stages : the first stage occurs in the nucleus where genome-size molecules are formed; the
second step takes place in the cytoplasm where the replicating molecules are large concatemers
(Goorha, 1982). This model also accounts for the earlier observations. Although the cytoplasm
was described as the site of virus assembly, morphogenesis and D N A synthesis (Granoff et al,,
1966; Bingen-Brendel et al., 1971 ; Kelly, 1975), some D N A was always replicated in the nucleus
(McAuslan & Smith, 1968; Aubertin et al., 1970; Goorha et al., 1978) and the necessity for a
functional nucleus was established (Goorha et al., 1977).
Based on the fact that most double-stranded D N A animal viruses have an arginine
requirement for completion of their growth cycle (Russell & Becker, 1968; Prage et al., 1968;
Spring et al., 1969; Obert et al., 1971 ; Archard & Williamson, 1971 ; Rouse & Schlesinger, 1972),
previous experiments showed that in FV 3-infected arginine-starved BHK cells, the D N A was
replicated and accumulated in the nucleus (Aubertin, 1975). Moreover, the functional
competence of a viral genome during the course of arginine starvation was recently demonstrated in the case of adenovirus (Auborn & Rouse, 1982). We have made use of these results to
investigate the fate of FV 3 D N A synthesized in the nucleus and to determine whether this
D N A can be packaged into virions,
Some viral proteins are selectively concentrated within the nucleus (Elliott & Kelly, 1980;
Martin et al., 198 l), and are likely to participate in the replication. Since these proteins may play
a role in the localization of the D N A in the various cell compartments, an analysis was made of
their synthesis and phosphorylation under arginine deficiency.
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0022-1317/84/0000-5915 $02.00 © 1984 SGM
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MARTIN AND OTHERS
METHODS
Cells and virus. Chinese hamster ovary (CHO) cells were grown in monolayers in alpha medium (Eurobio)
supplemented with 10~o calf serum. To deprive the cells of arginine, this medium was replaced 16 h before
infection by alpha medium devoid of arginine and supplemented with only 0.5~ dialysed calf serum.
FV 3 was produced and purified as described previously (Aubertin et al., 1973, 1980).
Infection conditions and labelling o f proteins, phosphoproteins and DNA. Cells were infected at a multiplicity of 40
p.f.u, of FV 3. After an hour's adsorption, the unbound virus was eliminated and the cells were incubated at 29 °C
in alpha medium with or without arginine. The D N A was labelled at different times following infection, usually
for 30 min, with 5 ~tCi [3H]thymidine (CEA, Saclay, France; sp. act. 25 Ci/mmol) per ml medium. Proteins were
labelled for the same period of time (or as indicated in the legend) with [3sS]methionine (Amersham International,
sp. act. 900 Ci/mmol) at a concentration of 12 ~tCi/ml medium. Phosphorylation of the polypeptides was followed
by the addition of 25 ~tCi [32p]orthophosphate (Amersham International) per ml phosphate-free medium.
Collection andfractionation o f cells. After the labelling or chase period, the cells were washed with phosphatebuffered saline (PBS) and scraped up in this buffer. They were fractionated following the method described
previously (Martin et al., 1981). When the samples were prepared for the determination of D N A polymerase
activity, the PBS buffer used contained neither MgCI2 nor CaC12.
Quantification o f DNA and protein syntheses. Protein synthesis and post-translational modification of proteins
were measured as the acid-insoluble material incorporated into the subcellular fractions. Aliquots were spotted
onto a Whatman 3MM paper square, immersed in 10~ trichloroacetic acid (TCA), heated in 10~ TCA in a
boiling water-bath and washed according to the method of Mans & Novelli (1961). The paper squares were dried
and the radioactivity associated was counted by liquid scintillation spectroscopy.
A similar procedure was followed for the determination of D N A synthesis, except that the TCA concentration
was 5 ~ and the heating step was omitted.
SDS-polyacrylamide gel electrophoresis. Denatured proteins were separated by electrophoresis using a
discontinuous buffer system (Laemmli, 1970) under the conditions described by Martin et al. (1981). To analyse
[35S]methionine-labelled proteins, the gels were treated and the fluorograms prepared as described by Bonner &
Laskey (1974). The phosphoproteins were revealed by direct autoradiography using an intensifying screen
(Cronex Lightning Plus, DuPont) (Laskey & Mills, 1977). Occasionally, to establish the ratio of [35S]methionine to
[32p]orthophosphate incorporated into a given polypeptide, the autoradiogram was used as a guide to localize the
molecule in the gel and the corresponding band excised from the dried gel. The pieces of gels were incubated in
500 ~tl H202 for 16 h at 37 °C, scintillation fluid was added and the radioactivity was measured.
Sedimentation in neutral sucrose gradients. To analyse the virus particles produced, the cytoplasmic fraction was
layered on a pre-formed linear (10 to 40~o, w/w) sucrose gradient prepared in 0.01 M-Tris-HC1 pH 8. Centrifugation was carried out for 1 h at 55000g in a Beckman SW27 rotor. Fractions of 1 ml were collected, an aliquot
of each was spotted onto a Whatman 3MM paper square, TCA-precipitated and the radioactivity was measured.
DNA analysis by sedimentation on alkaline sucrose gradients. The cell suspension, 105 cells in 1 ml buffer (0.15 MNaCI, 1.5 mM-KH:PO4, 2.5 mM-KC1, 1 mM-Na2HPO4, 0.5 mM-EDTA, pH 7.4), was loaded on top of a 30 ml
linear (5 to 20 ~ , w/w) sucrose gradient pre-formed over a 3 ml (65 ~ , w/w) sucrose cushion and overlaid with 2 ml
lysing solution (0.5 M-NaOH, 0.05 M-EDTA). The sucrose solutions were prepared in 0.3 M-NaOH, 10 mM-EDTA,
0.5 M-NaCI. The loaded gradients were kept overnight at 4 °C and then centrifuged at 72000g for 7 h. One ml
fractions were collected and the radioactivity was determined after TCA precipitation, the acid-insoluble material
being recovered on glass fibre filters (Whatman, GF/C).
DNA hybridization tofilter-bound F V 3 DNA. FV 3 D N A was immobilized on diazobenzyloxymethyl (DBM)paper using the commercially available paper supplied by Schleicher & Schiill. The activation step was performed
as recommended by the manufacturer. Paper squares were incubated for 30 rain in 30 ml 1.2 M-HC1 to which
0.85 ml NaNO2 at a concentration of 10 mg/ml had been added, and they were then washed twice for 1 min in
H20 and twice for 1 min with 20 mM-CH3COONa pH 4. Ten ~tg DNA, denatured by alkali treatment (15 min in
0.1 M-NaOH) and neutralized with 1.5 M-NaH~PO4, were spotted onto an activated paper square and allowed to
dry at room temperature. The filters were washed four times for 10 min in 0.5 M-NaOH and with 0.1 ~ SDS,
2 x SSPE (SSPE: 0.18 M-NaC1, 10mM-NaH2PO4, 1 M-EDTA, pH 7.7). Hybridizations were carried out in
Eppendorf tubes in the following mixture: 5 0 ~ deionized formamide, 1 × SSC (SSC: 0.15 M-NaCI, 0.015 Mtrisodium citrate), 0.02 ~ Ficoll, 0.02 ~ polyvinylpyrrolidone, 0.02 ~ bovine serum albumin, 0.01 M-sodium phosphate pH 6.8, 10 ~tg sonicated calf thymus DNA, 2 ~tg poly(A) and the labelled D N A to be analysed, in a final
volume of 300 ~tl.
Samples were incubated at 42 °C for 48 h. The papers were washed three times for 10 min with 5 × SSC,
similarly with 2 x SSC, 0.1 ~ SDS and then likewise with 2 × SSC at 60 °C. The filters were dried and the radioactivity was measured.
Protein kinase activity. The enzyme activity was measured as described previously (Martin et al., 1981) and
expressed as pmol of 32Pi transferred during a 15 min reaction with a lysate prepared from 2.5 x l0 s cells.
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F V 3 DNA in arginine-deprived cells
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Determination of DNA polymerase activity. Endogenous DNA polymerase activity was assayed for 30 min at
30 °C in an incubation medium (final volume, 1 ml)containing 1 mM-ATP,0.1 mM-dATP,0.1 mM-dCTP,0-1 mMdGTP, 0.01 mM-dTTP, 5 ~tCi [cc-32P]TTP(Amersham International; sp. act. 3000 Ci/mmol), 1 mM-dithiothreitol,
l mM-EGTA, 40 mM-HEPES pH 7.8, 100 mM-KCI, 5 mM-MgCI2, 0"5 mM-CaCI2 and 30 ~tl of the subcellular
fractions. The reaction was stopped by the addition of 20 mM-EDTA, 0.1~ SDS.
DNA synthesis was determined by measuring the incorporation of labelled nucleotide into acid-insoluble
material as described for DNA synthesized in vivo. The total DNA polymerase activity was determined with
activated calf thymus DNA (50 ~tg) added to the reaction mixture described above.
Electron microscopic autoradiography. To localize the newly synthesized DNA, the cells labelled with [3H]thymidine for 30 min (10 ~Ci/ml, sp. act. 25 Ci/mmol) were processed for electron microscopy as previously
described (Martin et al., 1981) and the treatment of samples for autoradiography was carried out according to
Larra & Droz (1970).
RESULTS
Influence o f arginine starvation on the site and the kinetics o f D N A replication
Non-confluent C H O cultures were pretreated overnight in alpha medium deficient in
arginine, then infected with FV 3 and incubated either in alpha medium deprived of arginine or
in complete medium. The newly synthesized D N A in infected cells was localized by pulse
labelling with [3H]thymidine for 30 min for different periods of time following infection and the
distribution in subcellular fractions, prepared as described in Methods, was determined. The
cytoplasm was separated into two fractions: a cytoplasmic fraction obtained after homogenization of the ceils in hypotonic buffer and centrifugation to eliminate the nuclei and
associated material, and a ' N P 4 0 fraction' resulting from the washing of the crude nuclear pellet
with the non-ionic detergent Nonidet P40. Under normal growth conditions, this NP40 fraction
is enriched in viroplasms containing viral D N A and proteins, whereas mainly DNase-resistant
material is recovered in the cytoplasmic fractions (Martin et al., 1981). The rate of D N A synthesis in the nuclear fraction increased with time and levelled off in both cases at about 7 h,
although arginine deficiency reduced that rate by a factor of two (Fig. 1 a). However, whereas
after 4 h the amount of newly synthesized D N A increased in the NP40 and cytoplasmic
fractions when cells were incubated in a normal medium, in the absence of arginine the quantity
of acid-insoluble 3H-labelled material recovered in these fractions remained very low and
represented less than 20~o of the total incorporation (Fig. 1 a).
The endogenous D N A polymerase activity (measured without the addition of template)
associated with the subcellular fractions after 6 h of infection was found to be higher in the
nuclei isolated from arginine-starved cells than from cells maintained in arginine-containing
medium (Fig. 1 b). The opposite situation was observed in the NP40 and cytoplasmic fractions,
the activities being very low for arginine-deprived cells. This was to be expected, as the amount
of viral D N A present in the cytoplasm is very small. The total D N A polymerase activity
measured after the addition of exogenous D N A was lower in arginine-starved cells (Fig. I c),
reflecting the reduced rate of protein synthesis; in the nuclear and NP40 fractions it was fourand threefold lower respectively. However, it is interesting to note that in the cytoplasm the
amounts of free D N A polymerase were almost identical whether the cells received arginine or
not. Thus, if D N A replication does not take place in the cytoplasm of arginine-deprived cells, it
can not be related to the absence of D N A polymerase in this cell compartment.
The localization of newly synthesized D N A was further supported by electron microscopic
autoradiographs. For this, cells infected and incubated in the appropriate medium were labelled
with [3H]thymidine, 30 min before processing for electron microscopy as indicated in Methods.
Typical distributions of the grains in the cells at 5 h post-infection are presented in Fig. 2. In the
majority of the infected cells maintained in arginine-free medium, grains were found predominantly in the nucleus (Fig. 2a), whilst for cells incubated in normal medium, they were
concentrated in cytoplasmic viroplasms (Fig. 2b).
The D N A , which is synthesized and remains concentrated in the nucleus under arginine
starvation, can reach the cytoplasm when optimal growth conditions are restored and the
medium is supplemented with arginine. Indeed, in cells pulse-labelled with [3H]thymidine for
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724
J.
P.
MARTIN
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Fig. 1. Influence of arginine starvation on the intracellular distribution of DNA and DNA polymerase
in FV 3-infected cells. Arginine-starved CHO cells were infected with 40 p.f.u./cell, then (a) incubated
in alpha medium with (O) or without (0) arginine. I"-1,Control culture. They were labelled for 30 rain
with [3H]thymidine at the time indicated after infection, collected, fractionated and DNA synthesis
determined as described in Methods. The DNA polymerase activity was measured in subcellular
fractions (N, nuclear; NP40, Nonidet P40; Cyt, cytoplasmic) prepared after 6 h infection, either
without (b) or with (c) the addition of exogenous DNA. The composition of the reaction mixture is given
in Methods and the incubation was for 20 min at 30 °C. Cells infected and maintained in complete
medium (Vl) or in arginine-minus medium (iN), and arginine-starved control cells (11) are shown.
30 min at 5 h post-infection and re-incubated after elimination of the radioactive precursor in
arginine-containing medium for 2 h, only 2 0 ~ of the labelled D N A was associated with the
nuclei; this value is close to the one obtained for the D N A synthesized in the presence of
arginine (Table 1). W h e n cells were kept in arginine-depleted medium, 7 5 ~ of the D N A
remained in the nuclear fraction after a 2 h chase, whereas immediately after labelling 7 7 ~ was
recovered in this fraction.
Compartmentalization of the newly synthesized D N A in the nucleus and its passage into the
cytoplasm is corroborated by the numbers of grains on electron microscopic autoradiographs.
As the cytoplasm is not totally free of grains, cells were identified as having D N A predominantly in the nucleus when more than 20 grains were found there and less than five in the
cytoplasm. T h i s was the case for over 5 0 ~ of the arginine-deprived cells when examined
immediately after a 30 min [3H]thymidine pulse (Table 1). Following a 2 h chase, in the presence
of arginine, only 30 ~ of the cells showed a nuclear concentration. The movement of the newly
synthesized D N A from the nucleus to the cytoplasm is thus controlled by an arginine-dependent
factor. Hybridization experiments of newly synthesized D N A with immobilized viral D N A
showed that 63 ~o and 84 ~ of the D N A produced, respectively, in arginine-starved cells and cells
kept in complete medium at 5 h post-infection, was of viral origin.
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F V 3 DNA in arginine-deprived cells
Fig. 2. Electron microscopic autoradiographs of DNA in FV 3-infected CHO cells, showing the intracellular localization of newly synthesized DNA, labelled with [3H]thymidine from 4.5 to 5 h postinfection, in cells incubated in arginine-depleted medium (a) or in complete growth medium (b). Bar
markers represent 1 ~tm.
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J. P. M A R T I N
AND
OTHERS
Table 1. Transfer to the cytoplasm of DNA replicated in the nuclei of F V 3-infected arginine-
deprived cells
Acid-insoluble
[3H]thymidine
(ct/min/1.5 x 105 cells)
Incubation
medium*
- Arg
+ Arg
-Arg
+ Arg
+ Arg
Time
after
infection
(h)
5
5
7
7
7
Total
cells
(T)
19241
33520
7420
10821
17726
Nuclear
fraction
(N)
14875
20190
5587
2170
2299
% N/T
77
60
75
20
13
Number of cells
with grains
Total
number
of ceils
examined
(T)
165
169
83
Predominant
nuclear
concentration
(N)
86
43
% N/T
52
22
26
31
* Cells incubated in arginine-plus ( + ) or -minus ( - ) medium were labelled, from 4-5 to 5 h after infection, with
[3H]thymidine. The distribution of the DNA was analysed immediately or at 7 h after a chase period in the same
medium or in medium supplemented with arginine, at 5 h (+Arg).
Size of DNA strands
To determine whether full-length copies of the viral genome were produced in the nucleus or
not, the sedimentation characteristics of D N A synthesized under arginine deficiency were
compared to D N A replicated under normal conditions. F o r this, two infected cultures
maintained in arginine-depleted or complete alpha medium were labelled with [3H]thymidine
for 30rain at 5 h post-infection and the cells collected immediately afterwards. Zonal
sedimentations were performed in alkaline sucrose gradients as described in Methods. To avoid
any breakdown of the D N A which can occur during the preparation of the nuclear fraction and
which has been observed in the first experiments (data not presented), the whole cells were
layered on top of the gradients. Although the majority of the labelled D N A migrated at the same
position as genomic D N A , a broad spectrum of the strand sizes was noticed, with a small
fraction of the molecules being longer than the genome (Fig. 3a). However, the distribution of
the D N A in the gradients was identical, irrespective of the infection conditions. To follow an
eventual conversion of this D N A , cells infected and labelled as above were re-incubated for 20 h
in the presence of an inhibitor of D N A synthesis, one culture being at that time supplemented
with arginine. Fig. 3 (b) shows a comparison of the D N A strand sizes. In all cases, a more homogeneous distribution of the D N A in the gradient could be observed; this distribution is
analogous to that of the virion D N A . This was also true for the cultures maintained under
arginine starvation, but the total amount of D N A recovered was significantly lower than that
present in cells where protein synthesis resumed after the addition of arginine. Thus,
considering that more than 80% of the D N A analysed in arginine-depleted cells was located in
the nucleus, it is clear that genome-size molecules are present in this cell compartment.
Functional FV 3 DNA is synthesized in the absence of arginine
Virus assembly did not occur under arginine starvation. However, after restoration of normal
medium, the growth cycle could be completed in the absence of additional D N A synthesis.
Two batches of arginine-starved C H O cultures were infected with F V 3 (m.o.i. 5 p.f.u/cell)
and incubated either in normal or in arginine-depleted medium. After 8 h, in some cultures
D N A synthesis was stopped by the addition of arabinosylcytosine (Ara-C) and, in the case of
arginine-starved cells, the medium was supplemented with arginine. The production of
infectious virus 24 h or 48 h later was measured by titration of the cell lysates on B H K cell monolayers (Table 2). Indeed, whilst in the cultures deprived of arginine there was no increase in the
virus titre from 8 h to 48 h post-infection, the addition of arginine allowed the formation of virus
particles and this took place in the absence of any new D N A synthesis. The amount of infectious
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FV 3 DNA in arginine-deprived cells
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Fig. 3. Alkaline sucrose gradient analysis of pulse-labelled DNA synthesized in FV 3-infected CHO
cells. The infected cultures maintained in complete alpha medium ( 0 ) or in arginine-minus medium
(©) were labelled for 4.5 to 5 h post-infection with [3H]thymidine and analysed immediately (a). After
the labelling period, some cultures were re-incubated in the presence of Ara-C (10 -3 M) either in the
same medium (normal medium, 0 ; arginine-minus medium, []) or after being supplemented with
arginine (O); they were analysed 20 h later (b). The vertical arrows indicate the position of virus
particle DNA analysed in parallel. Infection and sedimentation conditions were as indicated in
Methods. Sedimentation is from left to right.
Table 2. Production oJ infectious FV 3 particles affected by arginine starvation
Incubation
medium
+Arginine; at 8 h p.i.*
+Ara-C (10 3 M)
-Arginine; at 8 h p.i.
+Arg + Ara-C (10 3 M)
Infectious particles (p.f.u./ml)
at time post-infection (h)
c
z
~
0
8
24
48
3 x 105
8 x 10~
3 × 10 6
7 × 106
2-2 × 106 2.6 × 106
5 × 105
6.7 x 105
7.2 × 105
1.4 × 106
7 x 105
2.2 × t06
* Post-infection.
particles produced under these conditions after 48 h was almost identical to that o b t a i n e d in
cultures i n c u b a t e d in n o r m a l m e d i u m which received A r a - C at 8 h.
To ascertain that the infectious particles formed resulted from the e n c a p s i d a t i o n of the D N A
synthesized in arginine-depleted cells a n d not of parental D N A , [3H]thymidine was a d d e d to
arginine-starved cultures from 7 to 8 h post-infection; the labelled m e d i u m was then replaced by
the n o r m a l m e d i u m c o n t a i n i n g A r a - C a n d 24 h later the cytoplasmic fraction was analysed
by s e d i m e n t a t i o n on a sucrose gradient (under the conditions habitually used for virus
purification). W h e n the radioactivity was m e a s u r e d a n d the infectious particles present in the
g r a d i e n t fractions were titrated, a close correlation was found b e t w e e n the distribution of 3Hlabelled D N A and infectivity in the gradient (Fig. 4). Thus, it is clear that [3H]thymidine
incorporated into D N A u n d e r a r g i n i n e starvation is encapsidated into m a t u r e particles after rei n c u b a t i o n of the cultures in complete m e d i u m .
Protein synthesis under conditions of arginine depletion
The synthesis of F V 3 polypeptides is sequentially ordered a n d regulated (Willis et al., 1977;
Cordier, 1979; Elliott & Kelly, 1980; M a r t i n et al., 1982). To establish whether a lack of a r g i n i n e
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Fig. 4. Analysis of progeny particles formed by encapsidation of DNA synthesized under arginine
starvation. Arginine-depleted cells, infected for 7.5 h with FV 3 (m.o.i. 5 p.f.u./cell) were labelled with
[3H]thymidine for 30 min. The radioactive precursor was removed and the cells re-incubated in the
presence ofarginine and Ara-C (I0-3 M)for 20 h. A cytoplasmic fraction was prepared and analysed by
sedimentation on a neutral sucrose gradient as described in Methods. The radioactivity was measured
after TCA precipitation of aliquots of the gradient fractions ((3). Distribution of infectious virus
particles was followed by titration of the fractions on BHK cell monolayers and quantification of p.f.u.
(0). Sedimentation was from right to left.
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would significantly affect the production of some polypeptide species or merely result in a
reduced rate of synthesis for all of them, the proteins synthesized at different times after
infection and present in the three subcellular fractions were analysed. In the time interval of 2 to
8 h post-infection, some modifications could be noted. After 2 h the amount of [35S]methionine
incorporated in cells incubated in the absence of arginine was half that of cells incubated in the
presence of arginine; by 8 h the difference was more pronounced as the rate of synthesis
continued to decrease slightly in cells deprived of arginine while it doubled in normal cultures.
At the beginning of infection, 65, 20 and 15 % of the proteins were recovered respectively in the
cytoplasmic, NP40 and nuclear fractions (data not presented).
A similar distribution was found later in cells lacking arginine, whereas in its presence the
quantity of protein in the NP40 fraction increased with the time of infection, reaching 40yo of
the total by 8 h after infection. The percentage in the nuclear fraction remained close to 15. In
the absence of cytoplasmic D N A there was no viroplasm formation and the amount of viral
proteins in the NP40 fraction was therefore low. Electrophoretic analysis of the polypeptide
species synthesized shows clearly that the early groups (immediate early and delayed early) were
produced in the absence of arginine (Fig. 5). However, there were only traces of late polypeptides such as 48K and 15K, while polypeptides 70K, 63K or 12K were hardly detectable. The
intracellular distribution was similar to that found under normal conditions, polypeptides 90K,
54K, 42K and 31K being concentrated in the nucleus and polypeptide 16K in the cytoplasmic
fraction.
At 5 h, in the presence of arginine there is already a late pattern. The nuclear fraction
contained the polypeptides 90K, 70K, 63K, 31K, 12K at concentrations higher than in the cytoplasm while 58K, 56K, 16K were almost exclusively found in the cytoplasmic and NP40
fractions. After 8 h of infection, the total protein synthesis decreased in arginine-starved cells:
one early polypeptide, 31K, was more affected than the others.
Determination of protein kinase activity, a late function (Silberstein & August, 1976),
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i'~--I5
~ '--12
Fig. 5. Influence of arginine starvation on the polypeptide species synthesized in FV 3-infected cells.
Fluorogram of a 12.5~opolyacrylamide gel slab containing electrophoretically separated pulse-labelled
([3SS]methionine, 30 rain) protein in infected, arginine-starved CHO ceils (40 p.f.u./cell). Cells were reincubated after adsorption in the following: complete alpha medium ( + A), alpha medium devoid of
arginine ( - A ) ; arglnine-depleted medium until 5 h post-infection and then supplemented with
arginine ( - + A). The numbers give the time (h post-infection) of cell collection. Fractionation of the
cells was as indicated in Methods; the cytoplasmic fractions (Cyt) from 1.12 × l0 s cells, NP40 and
nuclear (N) fractions from 2-25 x 105 cells were analysed. The fluorograms presented were obtained
after an exposure of 1.5 days for the cytoplasmic fractions and 5 days for the NP40 and nuclear
fractions. The left column shows the structural polypeptides of FV 3, labelled with [3SS]methionine,
used as tool. wt. markers. The numbers in the margin indicate the polypeptide mol. wt. (x 10-3).
confirmed the strong reduction in the synthesis of group I I I polypeptides. Indeed, the respective
activities of lysates prepared with cells infected for 8 h and incubated in the presence or in the
absence of arginine were 0.75 and 0.15.
When the starved cultures were supplemented with arginine at 5 h and the proteins analysed
1 h afterwards, late species such as polypeptide 70K as well as polypeptides of tool. wt. higher
than 90K could already be found in the nucleus. A t 8 h, the profiles and the distribution were
almost identical to those observed in cells m a i n t a i n e d for the entire period in complete medium.
Phosphorylation of the polypeptides
The transfer of phosphate onto polypeptide chains m a y be a means of controlling their
biological activities, particularly in modifying their affinity for D N A (Wilcox et al., 1980).
Thus, as the synthesis of protein kinase was greatly depressed by arginine starvation, and
considering that this enzyme may be involved in the phosphorylation of the early polypeptide
species, the ratio of [3sS]methionine to [32P]orthophosphate incorporation into polypeptides was
measured between 1 h and 8 h after infection (Table 3). The data indicated that phosphorylation
was only slightly lower for polypeptides synthesized in the absence of arginine no matter what
their localization. To check whether individual polypeptides had been differentially affected, we
analysed polypeptides 90K and 31 K, two species found concentrated essentially in the nucleus
and known to be phosphate acceptors (Martin et al., 1981). The ratio of [3SS]methionine to
[32P]orthophosphate incorporated into the species present in the various cell compartments was
determined as described in Methods (Table 3). The consequence of arginine starvation was
more pronounced for the polypeptide 31 K present in the cytoplasm whereas in the nucleus only
small differences were observed.
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730
J. P. MARTIN AND OTHERS
Table 3. Relative phosphorylation of polypeptides in the different cell compartments
Polypeptides
Total proteins
Culture
medium
_+arginine
+
-
90K
31K
+
+
-
Ratio [3sS]methionine/[32P]orthophosphate
c
- "~
Cytoplasmic
NP40
Nuclear
fraction
fraction
fraction
22
14
5-7
28.4
13.8
6.8
20.2
9-4
6
25-6
11.6
5
23,5
20,3
8.3
32.5
13.8
9.7
It should be emphasized that the degree of phosphorylation of a given polypeptide can be
three- to fivefold higher in the nucleus than in the cytoplasmic fraction. This is true for polypeptides of apparent tool. wt. 90K and 31K, synthesized in cells maintained in complete or in
arginine-depleted medium.
DISCUSSION
Arginine starvation was used as a means of restricting FV 3 D N A replication to the nucleus.
Indeed, we have shown that approximately 80% of the newly synthesized D N A in infected,
arginine-deprived cells was found in the nucleus and remained there except when the medium
was supplemented with arginine. Under normal conditions, when the rate of D N A synthesis is
at its highest (7 to 9 h post-infection), only 40% of the newly replicated molecules are recovered
with the nuclear fraction.
Thus, according to the pattern proposed by Goorha (1982), stage 1 of D N A replication occurs
in the absence of arginine and the passage from stage 1 to stage 2 is greatly impaired but
probably not totally blocked.
Although it can not be excluded that a small fraction of the D N A recovered in the cytoplasmic
fraction may stem in part from a breakdown of some nuclei, electron microscopic autoradiography enabled grains to be found also in the cytoplasm.
Three conclusions can be drawn from the results presented here. (i) D N A replicated in the
nucleus of arginine-depleted cells is of genome size; (ii) the D N A synthesized under arginine
starvation is functional and, upon restoration of the normal growth conditions, can be
encapsidated to form infectious particles; (iii) D N A synthesis is restricted to the nucleus, most
likely because the expression of late genes is extremely limited.
The conclusion that genome-size molecules are present in the nucleus agrees with the recent
work of Goorha (1982). The observation of subgenomic size molecules reported earlier was
made after the isolation of the nuclei and a partial degradation may have occurred during cell
fractionation. The relatively heterogeneous distribution of the D N A in denaturing gradients
even after a chase is probably related to the structure of the genome, which contains singlestranded breaks in one D N A strand (Kelly & Avery, 1974).
The recovery of more than 50% of the D N A labelled during arginine starvation in particles
after an incubation in complete medium definitely indicates that nuclear D N A has been
encapsidated. Although D N A replicated in the nucleus is of unit length, it does not preclude the
hypothesis of an involvement of concatemeric molecules in D N A packaging. In fact, larger
precursor molecules can still be formed in the cytoplasm by recombination of unit size
molecules, an event which is likely to occur quite frequently between molecules presenting a
circular permutation and terminal redundancies (Goorha & Murti, 1982).
As to why FV 3 replication is impaired in arginine-starved cells, the data presented point to a
defect in the expression of the late genes. Early polypeptides are produced and they are distributed in the cell compartments as in the normal infection, but at 8 h post-infection, the synthesis
of polypeptide 31K is depressed. The degree of phosphorylation in the nucleus is only slightly
lower.
Some of these early polypeptides present in the nucleus must be involved in the replication of
viral DNA. In cells infected with herpes simplex virus and deprived of arginine, viral DNADownloaded from www.microbiologyresearch.org by
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F V 3 D N A in arginine-deprived cells
731
binding proteins involved in DNA replication could also be identified (Becker et al., 1980). In
parenthesis, it may be recalled that the phosphorylation of the two FV 3 polypeptides analysed,
90K and 31 K, was strikingly different in the various subcellular fractions under either restrictive
or optimal conditions. The properties of these molecules may therefore be modified depending
on their location in the cells. Numerous species of herpes simplex virus polypeptides are
phosphorylated but the extent of phosphorylation of the proteins accumulating in the nucleus is
not significantly different from that of their cytoplasmic counterparts (Wilcox et al., 1980).
However, in this case, the nucleus is the site of early events as well as of particle assembly.
It is clear that in FV 3-infected, arginine-starved cells, only traces of late polypeptide species
are synthesized, if at all. This is certainly not related to the difficulty of ensuring an efficient
polymerization of the amino acids in cells with a reduced pool of arginine, as the relative
abundance of early versus late species indicates that the block is specific to late polypeptides.
When added to normal medium, canavanine, an arginine analogue, did not interfere with the
synthesis of late polypeptides; therefore, it has been suggested that the presence of arginine at a
functional site of the molecules involved in the expression of late genes is unlikely (Elliott &
Kelly, 1980). However, addition of canavanine to arginine-depleted medium had a more
dramatic effect: it modified the regulation of the early species and did not allow the production
of late polypeptides (Cordier, 1979; Martin et al., 1981).
Whether or not the transcription of the late genes occurs remains to be established. In this
respect, it is interesting to make the comparison with vaccinia virus multiplication in argininedepleted cells. Although vaccinia D N A replication takes place, late m R N A s are not synthesized
(Obert et al., 1971) but transcription can resume upon the addition of ornithine or polyamines.
However, the presence of arginine is necessary for the production of infectious particles (Obert
et at., 1980).
The use of arginine deprivation has made it possible to differentiate the nuclear step of FV 3
replication from the later stage occurring in the cytoplasm. The recent description by Goorha
(1982), of a ts mutant, defective in this second stage, has provided the genetic evidence for a twostage replication. When data are available, a comparison of the genes expressed at the nonpermissive temperature in cells infected with the mutant with that of a wild-type under arginine
starvation may help to determine whether different events control the transition of FV 3 D N A
replication from the nucleus to the cytoplasm.
We thank G. Reinhardt, F. Lecerf and D, Spehner for excellent techniGal.assistance. This work was supported
by grants from the Institut National de la Sant6 et de la Recherche Mbdicale and from the D R E T (82-333).
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