Download Cell Division Does Not Affect Sendai Virus Genome Replication in

J. gen. Virol. (1984), 65, 2055 2060. Printedin Great Britain
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Key words: Sendai virus/persistently infected/cell division
Cell Division Does Not Affect Sendai Virus Genome Replication in
Persistently Infected BHK Cells
By L A U R E N T
ROUX*
AND P A S C A L E
BEFFY
Department o f Microbiology, 64 avenue de la Roseraie, University o f Geneva Medical School,
1205 Geneva, Switzerland
(Accepted 9 July 1984)
SUMMARY
The extent of Sendai virus genome replication in persistently infected B H K cells
actively growing or at confluence was followed by estimation of the [3H]uridine
incorporated into intracellular nucleocapsid RNA. First, we showed that, in the
presence of actinomycin D, actively growing persistently infected cells were taking up
threefold more [3 H]uridine than resting cells. This higher uptake exhibited by growing
cells was observed neither in persistently infected cells in the absence of actinomycin
D, nor in acutely infected cells in the presence of actinomycin D. Assuming that the
cellular pool of unlabelled uridine stays constant, we used a correction factor for this
difference in [3H]uridine uptake and estimated [3H]uridine incorporation in
nucleocapsid RNA, normalizing the data either to the amount of cell or of viral
template. Results showed that the viral genome replication, expressed either way, was
not significantly influenced by cell growth conditions.
Paramyxoviruses and rhabdoviruses are well known to be capable of establishing persistent
infections in their host cells in culture (Walker, 1968). While virus infections at high
multiplicities develop a cytopathic effect leading to cell death, in persistent infections virus
replication must be restricted in such a way that the cellular machinery is not disturbed by the
infection. Viral factors, such as homologous defective interfering (DI) particles or mutant
viruses, are known to exert such a restriction (in an as yet unknown way) during the
establishment and probably also later during maintenance of viral persistence (Holland et al.,
1980; Youngner & Preble, 1980). The question remains whether the host cell can participate in
modulating the extent of viral replication. For instance, it is known that, upon acute infection of
lymphocytes with measles virus, a member of the Paramyxoviridae, the stimulation of cells with
phytohaemagglutinin (PHA) greatly enhances the extent of viral production by 10000-fold
(Joseph et al., 1975). Similarly, the production of virus particles by a culture, called CAR 6,
persistently infected by the rhabdovirus, vesicular stomatitis virus (VSV) was shown to depend
on the growth conditions of the cells (Holland et al., 1978). Virus production ceased when CAR 6
cells were maintained in conditions minimal for cell division, i.e. no trypsinization but replacing
medium to maintain cell viability and remove dead cells. It resumed, however, when the cells
were stimulated to grow. These two examples suggested that virus replication was restricted in
resting cells and, therefore, that the cell growth conditions could modulate the extent of viral
replication. We decided to test this hypothesis using a B H K cell line (BHK-Pi) persistently
infected with Sendai virus, another member of the Paramyxoviridae family, 60 weeks after
establishment and measured the extent of viral genome replication in different cell growth
conditions.
BHK-Pi was established by co-infecting B H K ceils with a high multiplicity of standard (St)
Sendai virus mixed with DI particles (Roux & Holland, 1979). In the persistently infected
culture, close to 100% of the cells are infected at any time and, as in normal acute infection, all
the viral proteins are synthesized in the same relative molar ratios (Roux & Waldvogel, 1981,
1982). Viral genome replication takes place continuously with a constant excess of DI over St
0022-1317/84/00000-6140 $02.00 © 1984 SGM
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Fig. 1. Uptake of [3H]uridine by BHK-Pi cells in the presence of AMD. Samples of (b) confluent
(A259/PD > 4) or (a) actively growing (A259/PD < 2) BHK-Pi cells were labelled with 10 gCi/ml
[3H]uridine in the presence of 2 gg/ml AMD for various periods of time. At the end of the labelling
periods, the medium was discarded, the cells were washed twice with cold phosphate buffer and
collected in 1 ml 10~ TCA. After 30 min on ice, samples were centrifuged at 12000 r.p.m, for 15 min
and the supernatants were separated and used to estimate the TCA-soluble c.p.m. The pellets were
washed once with 10% TCA, once with ether, resuspended in 0.8 M-NaOH and incubated overnight at
37 °C Aliquots of the resuspended pellets were used to estimate (i) the TCA-insoluble c.p.m, by direct
liquid scintillation counting and (ii) the protein concentration of the cell material (Lowry et al., 1951).
Every time point was analysed in duplicate and the mean of the two values is shown: O - - O , total;
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C), TCA-soluble; O - - - O , TCA-insoluble.
Fig. 2. Uptake of [3H]uridine by BHK-Pi cells in the absence of AMD. As in the legend to Fig. 1,
except that AMD was omitted.
Fig. 3. Uptakeof[3H]uridinebyStvirus-infected BHKcellsinthepresenceofAMD. As in the legend
to Fig. 1, except that BHK-21 cells were acutely infected with St Sendai virus and labelled 18 to 24 h
post-infection in the presence of AMD.
genome replication (Roux & Holland, 1980). Although B H K - P i cells are morphologically
different from normal B H K (more epithelial-like than fibroblastic), they divide actively and are
repeatedly passaged by 1/4 to 1/6 dilution on alternate days, Since the Sendai viral genome is
always contained in a nucleocapsid structure very stable in high salt and therefore easily purified
by b a n d i n g in CsCI gradients (Kolakofsky, 1976), the extent of viral genome replication was
estimated by the incorporation of [3H]uridine into nucleocapsid R N A ( N C - R N A ) in the
presence of actinomycin D (AMD) which blocks the synthesis of all DNA-directed cellular
R N A . This estimation was made for 16 days using actively growing, or resting, cells. The degree
of cell growth was defined in relation to the cell density in a Petri dish. This density was
estimated by measuring the total amount of R N A present per 100 m m diam. Petri dish as
absorbance at 259 n m (A259/PD): this R N A was recovered in the pellets of the CsC1 gradients
used to purify the nucleocapsids (see above). Cells growing at a density lower than 2.5 A259/PD
were defined as growing, while those at a density higher than 2-5 A 2 s g / P D were defined as
confluent.
For the estimation of [3H]uridine incorporation into N C - R N A to be valid, one has first to
ensure that the specific activity of the 3H-labelled N C - R N A does not vary with the cell growth
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Table 1. Total uptake of[3H]uridine in the presence of AMD*
Growth
conditions
Growing
Confluent
Ratio: growing/confluent
Total [3H]uridine uptake in infected BHK cells
(c.p.m. x 10-5/mg protein) t
Expt. 1
Expt. 2
Expt. 3
~
~
BHK-St
BHK-Pi
BHK-St
BHK-Pi
BHK-St
BHK-Pi
2.55
2.04
3.6
1.99
6.85
5.75
1.92
0.75
2-46
0-83
5.43
1-03
(1.32)
(2.70)
(1-46)
(2.39)
(1-25)
(5-50)
* Standard virus (BHK-St) and persistently (BHK-Pi) infected BHK cells, actively growing or at confluence (for
definition, see text) were labelled with [3H]uridine and the total uptake by the cells was estimated after 6 h as
described in the legend to Fig. 1. Results obtained in three separate experiments are shown.
t ffa (mean of ratios from three experiments): BHK-Pi, 3.53; BHK-St, 1.34,
conditions. As the specific activity of the 3 H - R N A depends on the specific activity of the
[3H]uridine pool, and as this latter depends on the cellular uptake of [3H]uridine, it became
essential to follow the uptake of [3H]uridine in actively growing or confluent cells during the
labelling period used in our experiments. Fig. 1 shows that the uptake of [3 H]uridine by BHK-Pi
estimated in the presence of A M D varied with the cell growth conditions. Relative to the cell
mass, the amount of [3 H]uridine incorporated in the growing cells, found mainly as TCA-soluble
uridine because of the presence of the A M D , exceeded that found in confluent cells. Table 1
shows that the values of [3H]uridine uptake by the growing cells repeatedly exceeded those
measured in resting cells by a factor of 3 to 5. As shown in Fig. 2, this was not observed when
growing BHK-Pi cells were labelled in the absence of A M D (here most of the radioactivity was
found in TCA-insoluble counts since ribosomal R N A synthesis was no longer inhibited).
Moreover, it was not observed in B H K cells acutely infected with St virus, even in the presence
of A M D (Fig. 3 and Table 1). Therefore, the particular sensitivity to A M D in [3H]uridine
uptake only applied to BHK-Pi. Whether this peculiarity was triggered by persistent infection
remains an open question. It is however important for the correct interpretation of our data. If
one assumes that the pool of unlabelled intracellular uridine stays constant under different cell
growth conditions then the results imply that the specific activity of the uridine pool can change
and therefore that the efficiency of viral genome replication has to be corrected for the
difference in [3 H]uridine uptake. Therefore, a dividing factor of 3-53, calculated as the mean of
the differences in [3H]uridine uptake between growing and confluent cells observed in the three
experiments presented in Table 1 [(2-7 + 2.39 + 5.5)/3], was applied to all the values of
[3H]uridine incorporated in growing cells.
Incorporation of [aH]uridine was followed over 16 consecutive days by daily 6 h labelling
periods. The cells were passaged on days 1, 6, 9, 11 and 14. Fig. 4(a) presents a daily follow-up of
the cell densities in the Petri dishes: at days following cell passages (arrows), total AE59/PD was
low, increasing on the following day(s) until the next passage. Thus, the limit of 2.5 A259/PD,
chosen to discriminate between growing and confluent cells (see above), distinguished two
groups of cells. Group I comprised growing cells which consisted mainly of ceils analysed the
day following a passage, at days 2, 7, 8, 10, 12 and 15 (six values). Group II comprised confluent
cells analysed at days l, 3, 4, 5, 6, 9, 11, 13, 14 and 16 (10 values). This separation was not perfect,
since cells at day 3, classified as confluent, were clearly capable of further growth (increment of
A259/PD from 3 to 6, Fig. 4a). It was nevertheless kept, since 2.5 A259/PD conveniently
separated all the cell samples analysed before and after the passages. Moreover, inclusion of the
day 3 cell sample in group I would not have changed our conclusion.
When the incorporation of [aH]uridine into viral genome R N A ( N C - R N A ) was measured,
the values obtained showed some variation (Fig. 4b), with mean values (in) of [3H]uridine
incorporation of 0.95 and 0.92 3H c.p.m. × 10- 3/A259for growing and resting cells respectively.
Application of a M a n n - W h i t n e y test (Meddis, 1975) to the ranked values of the two groups
failed to show a statistical difference at the 1 ~ level of confidence using a one-tail test (P >> 0-5).
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Fig. 4. Daily estimation ofcell growth phase andofviralgenomereplicationinBHK-Pi. Everyday for
16 days, samples of BHK-Pi were labelled for 6 h with 10 gCi/ml [3H]uridine in the presence of 2 gg/ml
AMD. The cells were harvested, the intracellular nucleocapsids were purified on CsC1 density
gradients, and the total amount of RNA in the pellets of the CsCI gradients was measured. The
incorporation of [3H]uridine into NC-RNA was estimated as previously described (Roux & Waldvogel,
1981). (a) Amount of RNA present in the CsC1 pellets estimated as A259 (C)). (b) Incorporation of
[3H]uridine in NC-RNA per A259 during 6 h labelling periods of (O) confluent or (Q) actively growing
BHK-Pi cells. Arrows indicate days at which ceils were passaged.
Fig. 5. Daily estimation of viral genome template and of genome replication per template available.
Along with the estimation of [3H]uridine incorporation in NC-RNA presented in Fig. 4, aliquots of the
purified nucleocapsids were pelleted (300000g, 1 h, 4 °C); nucleocapsids were resuspended in gel
electrophoresis sample buffer (Laemmli, 1970) and electrophoresed on a discontinuous 12.5~
polyacrylamide gel. The Coomassie Brilliant Blue-stained gel was scanned in a Joyce-Loebl
densitometer to estimate the amount of NP in each NC sample (Roux & Waldvogel, 1981). (a) Amount
of nucleocapsid in the sample normalized to the amount of cell material (NCU). (b) Incorporation of
[3H]uridine as measured in Fig. 4(b) normalized to the amount of nucleocapsids in (O) confluent or ( 0 )
actively growing BHK-Pi. Arrows indicate days at which cells were passaged.
T h e r e f o r e , no a p p a r e n t influence of cell g r o w t h c o n d i t i o n s on viral g e n o m e replication was
o b s e r v e d in these experiments. It is n o t e w o r t h y t h a t w i t h o u t correction for the excess
[3H]uridine u p t a k e in g r o w i n g cells, the two groups o f values w o u l d h a v e b e e n significantly
different
(l~lgrowing = 3"36, mconnue,t = 0"92 3H c.p.m, x 10-a/A259, P = 0"01 ; d a t a not shown).
As the extent of viral g e n o m e r e p l i c a t i o n d e p e n d s on the availability of p o t e n t i a l t e m p l a t e ,
and as the a m o u n t of t e m p l a t e m a y v a r y in the cells due to c o n t i n u o u s cell division, it was of
interest also to m e a s u r e the a m o u n t o f intracellular nucleocapsids and to relate the a m o u n t o f
viral replication to the t e m p l a t e available. H e r e , the a m o u n t of viral n u c l e o c a p s i d was
d e t e r m i n e d as follows. A n aliquot o f n u c l e o c a p s i d purified on a CsC1 g r a d i e n t was c o n c e n t r a t e d
by c e n t r i f u g a t i o n and e l e c t r o p h o r e s e d on an S D S - p o l y a c r y l a m i d e gel. T h e C o o m a s s i e Brilliant
Blue-stained gel was scanned to e v a l u a t e the a m o u n t of N C protein ( N P ) present in the
nucleocapsid sample. T h e a m o u n t o f N P , directly p r o p o r t i o n a l to the a m o u n t o f viral g e n o m e
S h o r t communication
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R N A (St or DI RNA), was taken as the amount of nucleocapsid (NC). It was further divided by
the total A259 measured in the CsC1 gradient pellet from which the NC sample originated, and is
expressed as nucleocapsid units (NCU). N C U represented a normalization to the number of
cells so that different samples could be directly compared (see also Roux & Waldvogel, 1981).
Indeed, the amount of NC varied during the time of analysis: it slowly increased during the first
10 days of analysis, peaked at day 11, and then decreased for the next 5 days (Fig. 5 a). When the
efficiency of viral genome replication, as measured in Fig. 4(b) ( 3 H c.p.m, x 10-3/A259), was
normalized to the amount of nucleocapsid, and expressed as [3H]uridine incorporated into NCR N A per N C U (Fig. 5 b), again no significant difference between the two groups of values could
be found using a Mann-Whitney t e s t (l~9rowin 9 = 0-44, l~lconfluent = 0"46 3H c.p.m, x
10-3/NCU, P >> 0-5). In this case also, the uncorrected values showed a significant difference
between the two groups of cells (ffagrowin9= 1-56, ffaoonnuent= 0"46 3H c.p.m, x 10-3/NCU, P <
0.001; data not shown).
In conclusion, when the difference in [3H]uridine uptake observed between growing and
resting cells was taken into account, viral genome replication was not found to be influenced by
the cell growth conditions. This was the case whether genome replication was expressed as
[3H]uridine incorporated into N C - R N A per amount of cells, or per amount of template
available.
Our results do not necessarily contradict the data of Joseph et al. (1975) or Holland et al.
(1978), since we have looked at the level of viral genome replication while these authors
measured the production of viral particles. Taken together, these results might suggest that cell
growth conditions are likely to limit viral production by blocking a step in viral assembly rather
than by limiting viral genome replication. In view of reports showing that the cell surface
expression of viral antigens is cell growth-dependent (Ehrnst, 1979; Roux & Waldvogel, 1983),
we would suggest that, in resting cells, the insertion of the viral membrane antigens in the cell
plasma membrane, a step which is a prerequisite for the budding of viral particles, may be
restricted in such a way that maturation of viral particles is prevented. One could also argue that
a study following viral genome replication in persistently infected cells mainly reflects
estimation of DI genome replication (Roux & Holland, 1980). Therefore, a possible inhibition of
standard virus genome replication in resting cells could have been missed. Such a selective
inhibition could in turn affect viral particle production by limiting transcription and viral
protein synthesis. This is unlikely, however, since DI genome replication depends on viral
protein synthesis as well. Therefore, inhibition of protein synthesis, due to lack of template
available for transcription, would have affected DI genome replication in our experiments.
This work was supported by grant No. 3. 3324). 82 from the Swiss National Research Foundation. The authors
wish to thank Benjamin Blumberg for critical reading of the manuscript.
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Short communication
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