J. gen. Virol. (I98O), 47, 373-384
373
P r i n t e d in Great Britain
Inhibition of R N A and Protein Synthesis in Interferon-treated
HeLa Cells Infected with Vesicular Stomatitis Virus
By M A R C E L L A S I M I L I , F R A N C E S C A DE F E R R A
AND C O R R A D O B A G L I O N I
Department o f Biological Sciences, State University o f New York at Albany,
Albany, New York 12222, U.S.A.
(Accepted 6 November 1979)
SUMMARY
Synthesis of vesicular stomatitis virus RNA and protein is almost completely
inhibited in HeLa cells treated with relatively high doses of human fibroblast
interferon. With lower concentrations of interferon virus replication is inhibited,
but near normal amounts of virus RNA are found in cells infected at a m.o.i, of
Io. All the virus RNA species are found in these cells with the exception of
genomic size RNA. In contrast, synthesis of all the virus proteins is equally
inhibited in proportion to the interferon concentration used to treat the cells.
This inhibition is due to a decline in the rate of protein synthesis, which occurs in
interferon-treated cells sooner after infection than in untreated cells. The decreased
rate of protein synthesis is accompanied by a change in the polysome pattern of
infected cells, characterized by polysome run-off and increase in 8oS ribosomes.
At the same time, a larger proportion of the virus poly(A)-containing RNA is
not associated with polysomes in interferon-treated cells than in control cells.
The non-polysomal virus RNA has a sedimentation rate identical with that of
polysomal virus RNA. Possible causes for the decline in the rate of protein
synthesis observed in interferon-treated cells are discussed.
INTRODUCTION
In interferon-treated cells infected by vesicular stomatitis virus (VSV), accumulation of
virus transcription products is reduced (Marcus et al. I97I; Manders et al. ~97z; Repik
et al. I974; Baxt et al. I977; Marcus & Sekellick, I978; Thacore, 1978), synthesis of virus
proteins is decreased (Baxt et al. I977; Thacore, 1978) and virus replication is inhibited.
The mechanisms involved have not yet been clarified. By infecting Vero cells with a ts
mutant of ¥SV at a non-permissive temperature, Marcus & Sekellick (1978) showed that
the rate of primary transcription is reduced by interferon treatment. Primary transcription
is carried out by virion-associated transcriptase even in the absence of virus protein synthesis, as for example in cells infected in the presence of inhibitors of protein synthesis.
Marcus et al. (I971) and Manders et al. (I972) had previously shown that VSV primary
transcription is inhibited in interferon-treated cells in the presence of cycloheximide. Any
reduction in the accumulation of primary transcripts in a negative-strand virus-like VSV,
would result in a marked reduction in virus protein synthesis. Baxt et al. 0977) reported,
however, that near normal amounts of virus RNA are present in interferon-treated monkey
and human cells infected with VSV; only those virus RNA species, the synthesis of which is
dependent on virus protein synthesis, were found in much reduced amounts. These results
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M. S I M I L I , F. DE F E R R A A N D C. B A G L I O N I
are consistent with an inhibition of virus protein synthesis in interferon-treated cells
(Baxt et al. J977). In agreement with these observations, Thacore (I978) reported that in
a simian cell line interferon did not inhibit VSV primary transcription though virus protein
synthesis was completely inhibited. The action of interferon on virus transcription a n d / o r
translation may therefore depend to a large extent on the particular cell line studied
(Thacore, 1978).
We have examined virus R N A and protein synthesis in HeLa ceils treated with human
fibroblast interferon and infected with VSV. Our purpose was to define the characteristics
of the inhibition of virus R N A accumulation and protein synthesis in this system. We have
obtained evidence for both a reduced rate of accumulation of virus transcripts and inhibition
of protein synthesis in interferon-treated cells to an extent dependent on the m.o.i, and
on the dose of interferon.
METHODS
Cells. H e L a cells were grown in suspension cultures in Joklik's modified minimal essential
medium (MEM) with 5 % horse serum. Cells in logarithmic growth were collected at about
5 × 1o5/m 1, concentrated by centrifugation and resuspended in fresh medium for virus
infection.
Virus growth and assays. Wild type VSV (Indiana serotype) was a gift of Dr McSharry,
Department of Microbiology, Albany Medical College. The virus was grown in HeLa
cells, washed with serum-free MEM and resuspended at 1o7/rnl in MEM containing
I4 rnM-N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid ( H e p e s ) - K O H buffer, pH7'2,
and 2 mM-glutarnine. VSV was added for 45 rain at 30 °C at a m.o.i, ofo.ol to prepare seed
stocks of virus, or at a rn.o.i, of I to prepare high titre VSV stocks. Ten volumes of the same
medium with 1o % foetal calf serum were then added, together with I /~g/ml of actinornycin
D (Banerjee & Rhodes, 1973). The cells were incubated for 20 h at 37 °C, frozen and
thawed three times and centrifuged at 3 o o o o g for to rain. The supernatant from cells
infected at a m.o.i, of I constituted a standard virus stock preparation, which contained
4 to 8 × IOs p.f.u./ml. The virus titre was determined by plaque assay on H e L a cells.
Cells (~'5 ml at I-2 x ioT/rnl) were infected with o'I rnl of suitably diluted virus stock and
then mixed with I ml of I'3 % Noble agar (DIFCO) dissolved in Earle's salts solution and
kept at 43 °C. The cell suspension was poured over io ml of I % agar in F-I3 medium
(GIBCO) with i o % foetal calf serum in lo crn diam. tissue culture dishes. These were
incubated for 2 to 3 days in 5 % CO2 before counting plaques.
Interferon treatment and assay. H u m a n fibroblast interferon (3 × Io 5 reference units/rag
of protein) was obtained from the Interferon Working Group of the National Cancer
Institute, N I H , Bethesda, Md. HeLa cells were treated with the indicated interferon concentrations for I7 h before infection with VSV. The antiviral effect of interferon was
assessed by plaque assay and by reduction of infectious virus yield. The plaque assay was
carried out as described above with interferon-treated and control cells, and virus dilutions
which gave between ~oo and 2o0 plaques per dish with control cells. The yield reduction
assay was carried out by treating cells with different interferon concentrations and infecting
them at a m.o.i, of Io. Eight h p.i. the cells were broken by freezing and thawing. The
clarified (3oooog for Io min) supernatant was assayed by the plaque method to determine
the virus yield.
R N A synthesis. One ml of cells infected as described above was incubated for 2o rain with
5/zg of actinornycin D and then for 30 min with ro/~Ci of ZH-uridine (20 to 40 Ci/rnmol).
The cells were collected by centrifugation, washed twice with phosphate buffered saline at
o °C and lysed in 0"5 ml of SDS-buffer (o.I M-NaCI, t mM-EDTA, o'5 % sodium dodecyl
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Interferon action on R N A and protein synthesis
375
sulphate and lo mM-tris-HCl, pH 7"5). A sample was precipitated with an equal vol. of
2o% trichloroacetic acid and collected on Whatman G F / A filters for counting. The
remainder was applied to I5 to 3o% sucrose gradients in SDS-buffer and centrifuged as
indicated in the figure legends.
Protein synthesis. One ml of cells infected at a m.o.i, of To was collected by centrifugation
and resuspended in medium prepared with dialysed horse serum, lacking both methionine
and Iysine. After Io rain at 37 °C, to/~Ci of either asS-methionine or aH-lysine were added.
The ceils incubated with 3~S-methionine were diluted with IO ml of ice-cold phosphate
buffered saline, collected by centrifugation and lysed in o.z ml of lo mM-KC1, I"5 myrmagnesium acetate, I mM-dithiothreitol, 2o myr-Hepes-KOH buffer, pH 7"4, and 1%
Triton X-too. The supernatant fluid obtained by centrifuging for 5 min at 3oooog was
used for gel electrophoresis analysis. A o.I ml sample of cells incubated with aH-lysine
was directly spotted on Whatman 3MM papers filters and counted as previously described
(Weber et al. I975).
Gel electrophoresis. Samples (3o/zl) from cells incubated with ~sS-methionine were fractionated on I2"5 % polyacrylamide gels according to Laemmli (I97O). The dried gels were
exposed to pre-fogged X-ray films and scanned through a recording microdensitometer
(Laskey & Mills, I975). The areas under peaks of virus proteins were determined and
expressed in arbitrary units. Control experiments were carried out with different amounts
of labelled proteins and different times of exposure of the autoradiographs to obtain a linear
relationship between the amount of radioactive protein and the units of peak areas.
Polysome analysis and isolation of poly(A)-containing RNA. Cell extracts were prepared
from 2oo ml of cells infected as described above, collected by centrifugation, washed and
homogenized as previously described (Weber et al. r975). Cell extract (o'3 ml) was applied
to 1o to 4o % sucrose gradients in IO mM-NaCI, I'5 mM-MgC12 and IO mM-tris-HCl, pH 7"5,
and centrifuged for 9o rain at 2ooooo g in the SW4I rotor. The A 2G0was determined with a
recording spectrophotometer. To isolate poly(A)-containing RNA, the cell extracts were
fractionated b y a modification of the method of Rose & Lodish (I976). Samples of o'3 ml
of cell extract were adjusted to o'5 M-NaC1 and 3o raM-magnesium acetate in a final vol.
o f o ' 4 ml and layered on 4"5 ml I5 to 3o% sucrose gradients in the same salts with 20 mMH e p e s - K O H buffer, pH7"4. The gradients were centrifuged for 3 h at 24oooog in the
SW5o. I rotor. Ten fractions were collected through a recording spectrophotometer and the
bottom eight fractions containing ribosomal subunits (8oS ribosomes are dissociated into
ribosomal subunits under these conditions) were combined and used to measure A~60 and
poly(A)-containing RNA not bound to polysomes. The pellet containing polysomes was
dissolved in I ml of SDS-buffer to determine A260. An equal vol. of 1% sodium dodecyl
sulphate was added to the gradient fractions, and T/2o or z / t o vol. of 5 M-LiC1 was then
added to these fractions and to the re-dissolved polysome pellet respectively. The samples
were heated to 6o °C for I rain and immediately chromatographed on 0"5 × 0"4 cm columns
of oligo(dT)-cellulose as previously described (Weber et aL 1979). A sample of the poly(A)containing RNA was counted, while the remainder was analysed on sucrose gradients.
Experimental conditions. Unless otherwise stated HeLa cells were pre-treated for I7 h
with the desired concentration of interferon and infected with VSV at a m.o.i, of Io.
RESULTS
When the antiviral effect of human fibroblast interferon was evaluated in VSV-infected
HeLa cells by a plaque reduction test and by a yield reduction assay, the results were in
good agreement (Table l). From these data it can be calculated that about 35 units of the
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M. S I M I L I ,
F. DE F E R R A A N D C. B A G L I O N I
Table I. Effect of interferon concentration on vesicular stomatitis virus replication and virus
RNA synthesis*
Interferon
concentration
(units/ml)
Plaque reductiont
( ~ of untreated)
2o
4o
80
zoo
34
6I
9o
95
Yield reduction++ RNA synthesis§
( ~ of untreated) ( ~ inhibition)
36
46
72
n.d.
3
I4
42
n.d.
* For details of the experimental procedures see Methods.
t Plaque reduction: mean from several determinations with a VSV input giving IOO to 2oo plaques per
plate with untreated cells.
++ The yield of virus was measured after infection at a m.o.i, of Io.
§ Virus RNA synthesis was measured between I and 8"5 h p.i. at the same m.o.i. Infected cultures were
labelled with ~H-uridine; the RNA synthesis relative to VSV-infected untreated cells was used to calculate
~o inhibition (Ioo - ct/min interferon-treated/ct/min untreated x Ioo).
human interferon used are equivalent to one PRs0 (VSV) unit measured in fibroblasts,
indicating that: (i) HeLa cells are less responsive to the interferon used than fibroblasts,
and (ii) the effective dose of interferon is about 35-fold less than the units shown in legends
and text.
In preliminary experiments HeLa cells were treated before VSV infection with interferon
concentrations higher than ioo units/ml. VSV replication was effectively suppressed by
this treatment, and virus RNA and protein synthesis were almost undetectable (data
not shown). For the following experiments, therefore, the cells were treated with interferon
concentrations which, while effective in reducing the yield of infectious virus, still allowed
substantial virus RNA and protein synthesis (Table I and Fig. I to 3).
RNA synthesis
Virus RNA synthesis was measured in VSV-infected cells by incubating the cells for
30 rain periods with 3H-uridine after inhibition of host RNA synthesis with actinomycin D
(see Methods). In control cells infected at a m.o.i, of I the peak of virus RNA synthesis
occurred at about 5 h p.i., whereas in cells infected at a m.o.i, of 5 or ro the peak of virus
RNA synthesis occurred at about 3"5 h p.i. (Fig. t). In interferon-treated cells, synthesis of
virus RNA was markedly inhibited. At a m.o.i, of 1 or 5, virus RNA synthesis was considerably delayed and occurred over a longer time period without reaching a well-defined
peak (Fig. a a and b); at a m.o.i, of l o, there was a well-defined peak of synthesis which was
delayed about ~ to 2 h relative to untreated cells. The total amount of virus RNA synthesized
at this m.o.i, was reduced in proportion to the interferon concentration used to treat the
cells (Table I).
The R N A synthesized by interferon-treated and control cells infected at a m.o.i, of Io
was further characterized by sucrose gradient sedimentation. The patterns of the virus RNA
synthesized at 4 to 4"5 and 5 to 5"5 h p.i. are shown in Fig. 2. The virus R N A sedimented
in three peaks corresponding to genomic size RNA (38S, peak 1), L protein mRNA (28S,
peak 2) and m R N A for the other four VSV proteins 02S to 16S, peak 3). In addition, some
small tool. wt. RNA sedimented near the top of the gradients. The amount of virus RNA
synthesized between 4 and 4"5 h was similar in interferon-treated and control cells, whereas
at 5 to 5"5 h RNA synthesis was greater in interferon-treated cells because their peak of
virus RNA synthesis is delayed (Fig. ac). Virus RNA species of identical sedimentation
value were synthesized by interferon-treated and control cells, except that interferon-treated
cells did not synthesize appreciable amounts of genomic size RNA even at the peak of
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Interferon action on R N A and protein synthesis
377
Q
2.0
(a)
/"-...
m.o.l. = 1
/\
1.5
1.0
/
/
o-5
• ~
/'/.
~"'=
.~
#
V
2.0 ~- (b)
m.o.i. = 5
x 1.5
.E
~ 1.0
4 '
(c)
ln.o.i. = 10
2
4
6
Time p.i. (h)
8
Fig. I. Synthesis of virus RNA in interferon-treated and control HeLa cells infected with different
amounts of VSV. Control cells (O--Q) and cells treated with 4o (11- -II) and 8o (A- - -A) units/
ml of human fibroblast interferon were infected at the indicated m.o.i. Virus RNA synthesis was
monitored as described in Methods. Abscissa: time p.i.; ordinate: the ct/min of 3H-uridine
incorporated in 3o min pulses.
R N A synthesis (Fig. 2e and f ) , or later (data not shown). Such a failure to synthesize 38S
R N A has previously been observed (Baxt et al. 1977). We also observed a slight reduction
in the synthesis of 28S virus R N A in cells treated with 80 units/ml of interferon (Fig. 2c
and f).
Protein synthesis
Virus protein synthesis was next investigated in cells treated with different interferon
concentrations and infected at a m.o.i, of IO. Cells were labelled with 35S-methionine at
different times after infection and the labelled polypeptides were fractionated by gel electrophoresis (Fig. 3). The pattern of virus protein synthesis was the same in untreated VSVinfected cells and in cells treated with 2o to 8o units/ml of interferon before infection, but
the amounts of the virus proteins synthesized decreased with the interferon concentration
used. Virus protein synthesis was also estimated by scanning gel autoradiographs (Fig. 4).
The peak areas of three VSV proteins synthesized throughout the first 5"5 h of infection
were determined; similar data were obtained for the other two virus proteins (N and L),
not shown in this figure.
The virus proteins were found in similar relative ratios in VSV-infected cells whether
untreated or treated with different interferon concentrations. The rate of accumulation of
virus proteins, however, decreased with the interferon concentration and there was three to
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M. S I M I L 1 ,
F. D E F E R R A
Control
3
AND
C. B A G L I O N I
Interferon-treated
40 units/ml
80 units
(a)
2 8 S 18S
'
.A
,
"
-
I
J
Ik
J
I
I
3-
I
I"
I
I
,
, .
20
11
2_
- . , .i 4 . ,
I
5
10
15
20
5
,
10 15
Fractions
20
5
10
15
Fig. 2. Sedimentation analysis o f the virus R N A synthesized by interferon-treated a n d control
H e L a cells infected with VSV. Control cells a n d cells treated with 40 to 8o u n i t s / m l of interferon
were infected at a m.o.i, of lO a n d incubated for 3o m i n with 3H-uridine at (a) 4 a n d (b) 5 h p.i.
T h e R N A was fractionated by sucrose gradient centrifugation for 17 h at 7oooo g a n d precipitated
with 5 ~ trichloroacetic acid for counting. T h e positions o f m a r k e r ISS a n d 28S r i b o s o m a l R N A s
are indicated by arrows.
Interferon used for pre-treatment (units/ml)
Time
pulsed
c
(h) ...... 2
0
~"
3
~
4
5
•
20
~
2
3
4
5
•
f
80
A,
2
3
5
--L
--G
--NS
--N
--M
Fig. 3. Electrophoretic analysis of the proteins synthesized after VSV infection in control H e L a
cells a n d in cells pre-treated with interferon at the indicated concentrations. T h e cells were pulsed
for 3o m i n with 3~S-methionine at the times indicated. F o r other details, see M e t h o d s . T h e n u m b e r s
on the left indicate three unidentified cellular proteins which are well separated f r o m virus
proteins. T h e letters on the right indicate the VSV proteins.
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Interferon action on R N A and protein synthesis
379
15
10
10
5
e~
.
G
I0-
2
3
4
Time p.i. (h)
5
Fig. 4. Synthesis of VSV proteins in control cells ( m - - m ) and in cells treated for I7 h with zo
( 0 - - 0 ) , 4o ( A - - l , ) and 8o ( T - - T ) units/ml of interferon. The peak area was determined from
scans of autoradiographs (see Fig. 3) and is expressed in arbitrary units. The cells were pulsed for
3o min with asS-methionine at the indicated times after infection. For details of the analysis see
legend of Fig. 3-
(b)
(a)
t
15
0
x 10
gs
/
3
t
I
I
4
5
6
I
I
3
7 2
Time p.i. (h)
I
I
i
I
4
5
6
7
Fig. 5. Time course of protein synthesis in control cells and in cells treated with interferon and
infected with VSV. (a) Control (11--11) and cells treated with 8o units of interferon ( y ~').
(b) Control infected cells ( I I - - I ) and cells treated with 40 ( A - - I , ) and 8o ( V - - T ) units/ml of
interferon before infection. The cells were pulse labelled with 3H-lysine for 1 h at the times shown
(see Methods). The data obtained have been plotted as the cumulative amounts of protein synthesized in order to show the change in rate with time p.i. (no change was observed in the first 2 h p.i.).
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M. S I M I L I ,
F. D E F E R R A
AND
C. B A G L I O N I
Table 2. Partitioning of ribosomes and poly(A)-containing mRNA *
Cell t r e a t m e n t
r
Interferon
(units/ml)
O
40
O
4O
% of ribosomes
% labelled poly(A)VSV-infection in polysomes containing RNA in polysomes
--+
+
62
5Z
66
38
95
94
73
5O
* T h e cells were infected at a m.o.i, of I0 a n d labelled between 2 a n d 5 h p.i. with I0 # C i / m l of 3Huridine. In the experiments where VSV infection was omitted, the cells were labelled for 2 h with no actinom y c i n D added. T h e figures represent averages of duplicate experiments. See M e t h o d s for the fractionation
of polysomes a n d the isolation o f poly(A)-containing R N A .
four times less virus protein in cells treated with 8o units/ml of interferon than in untreated
infected cells. This difference in the accumulation of virus protein most likely results from
a different rate of synthesis and, even though we cannot exclude that turnover of virus
proteins may also play some role, we will, for convenience, use the term 'synthesis' in the
following sections.
It is important to note that the reduction in virus protein synthesis in interferon-treated
ceils is not accompanied by an increased synthesis of cellular proteins. The pattern of
proteins synthesized in uninfected cells and in interferon-treated uninfected cells was
identical (data not shown). A few clearly-defined bands can be distinguished above the
high background due to the complex pattern of cellular protein synthesis. Three of these
bands could be followed in infected cells, because they are well resolved from virus proteins
(Fig. 3). They were uniformly diminished in both untreated and interferon-treated infected
cells, regardless of the relative amount of virus protein synthesis. Therefore, there is a
shut-off of host protein synthesis in cells treated with 2o to 8o units/ml of interferon and
infected with VSV but, at the same time, synthesis of virus proteins is markedly inhibited.
Protein synthesis was also measured by determining 3H-lysine incorporation. In untreated
and interferon-treated uninfected cells, protein synthesis proceeded linearly and at about the
same rate (Fig. 5a). In VSV-infected cells, protein synthesis slowed down at about 5 h p.i.,
and in interferon-treated cells the rate of protein synthesis decreased even further, proportionally to the interferon concentration (Fig. 5 b). This is in accordance with the observations that virus protein synthesis is reduced in these cells and that at the same time host
protein synthesis is shut off. The overall rate of protein synthesis in interferon-treated
cells declined earlier after infection than in untreated cells.
Polysome pattern
This observation was confirmed by examining the polysome patterns (Fig. 6). Fewer
polysomes and more 8oS ribosomes were present in interferon-treated infected cells at 5 h
p.i. than in the other cells, perhaps because of an imbalance between the rate of polypeptide
chain initiation and that of elongation (see Lodish, I976). Since predominantly VSV
proteins are synthesized at this time (Fig. 3), the polysome pattern reflects the relative rate
of initiation and elongation of virus polypeptides.
The results presented are representative of repeated experiments. The same pattern of
polysome run-off was observed in cells treated with 4o units/ml of interferon and infected
with VSV. The distribution of ribosomes between polysomes and the non-polysomal fraction was also determined by a different procedure, which allowed a quantitative measurement of ribosomes as indicated in Table 2. The results were in agreement with those obtained
by scanning sucrose gradients. This procedure was also used to prepare poly(A)-containing
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Interferon action on R N A and protein syn!hesis
Control cells
38I
Interferon-treated
cells
0.4-
0.2
(b)
0-6
B
0.4
0-2 - - ~
~
Fig. 6. Polysome profiles. Interferon treatment was with 80 units/ml. Cell extracts were prepared
and, after centrifugation, analysed through a recording spectrophotometer as described in
Methods. (a) Not infected; (b) VSV-infected for 5 h.
RNA from the polysomal and non-polysomal fractions. The relative amount of virus
poly(A)-containing RNA associated with polysomes in untreated and interferon-treated
cells, and of cellular RNA in uninfected cells was determined by chromatography on oligo(dT)-cellulose as described in Methods (Table 2). About 95 % of the poly(A)-containing
RNA was found associated with polysomes in uninfected cells. In cells infected with VSV
approx. 65 to 8o % of the virus RNA was found associated with polysomes. In cells treated
with interferon and then infected with VSV, the proportion of virus RNA associated with
polysomes was reduced to 4o to 6o% of the total virus poly(A)-containing RNA. The
decrease in the relative amount of ribosomes present in polysomes correlated with an
increased amount of virus RNA not associated with polysomes.
This was further confirmed by analysing the RNA prepared from the polysomal and nonpolysomal fractions of interferon-treated cells. The cells were pulsed with aH-uridine from
z to 5 h p.i. and the RNA analysed by sucrose gradient sedimentation to display the I2 to
16S RNA coding for four of the VSV proteins (Fig. 7)- Synthesis of virus RNA was reduced
in interferon-treated cells during this labelling time (see Fig. 2), but the sedimentation
pattern of RNA associated with polysomes was indistinguishable from that of the nonpolysomal fraction. The virus RNA from interferon-treated cells sedimented identically to
RNA from untreated infected cells, though the ratio of polysomal to non-polysomal RNA
was quite different in the two samples analysed. We concluded that treatment with interferon does not appreciably change the size of the virus mRNAs in infected cells, confirming
the previous results of Manders et al. (197z), Baxt et al. (I977) and Marcus & Sekellick
(I978), but alters their association with ribosomes by mechanism(s) which have not yet
been elucidated.
DISCUSSION
In interferon-treated HeLa cells infected with VSV, the accumulation of virus transcription and translation products is inhibited in a characteristic way. At a m.o.i, of i and at
relatively high interferon doses, the accumulation of transcription products is drastically
reduced, whereas at a m.o.i, of 5 to io and at lower interferon doses virus transcripts are
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M. S I M I L I ,
F. D E F E R R A
AND
C. B A G L I O N I
24
20
16
12
O
×
8
~
4
e-,
~e)
1
(a)
5
10
15
Fraction
Fig. 7. Sedimentation analysis of poly(A)-containing RNA obtained from the polysomal (Q--O)
and non-polysomal (11--11) fraction of VSV-infected cells: (a) control and (b) treated with 40 units/
ml of interferon. The cells were labelled with ~H-uridine from 2 to 5 h p.i. ; the polysomal and nonpolysomal fractions were resolved as described in Methods and the poly(A)-containing RNA prepared and centrifuged for I7 h at 14oooog. The ~ of total labelled RNA bound to polysomes was
8o ~ in (a) and 6o ~ in (b).
found in near normal amounts. However, R N A is accumulated at a slower rate relative to
control cells.
It seems likely that the inhibition of primary transcription reported by Marcus et al.
0 9 7 0 , Manders et al. (2972) and Marcus & Sekellick 0978) may account for the reduction
by interferon in the accumulation of virus transcripts at low m.o.i. Inhibition of primary
transcription would result in reduced synthesis of virus proteins and could effectively block
the amplification of virus R N A synthesis and production of new virus. This effect on primary
transcription, however, is not sufficient to block amplification in cells infected at a higher
m.o.i. As previously observed by Baxt et al. (1977) and by Thacore 0978), virus transcription products are accumulated in some cell lines in near normal amounts, but production
of infectious virus is effectively inhibited, indicating that antiviral mechanisms other than
the inhibition Of primary transcription may be involved in reducing virus yield.
In agreement with B a x t e t al. (I 977) and Thacore (1978), we find that synthesis of VSV
proteins is reduced even when near normal amounts of virus m R N A s are produced.
Therefore, synthesis of genomic size RNA, which is dependent on the synthesis of virus
protein, is drastically reduced in interferon-treated cells. Presumably, sufficient amounts
of virus protein have to accumulate before replication of genomic size R N A and virus
assembly can proceed. For example, the M protein has a regulatory function on transcription: group I I I t s mutants of VSV synthesize an altered M protein at non-permissive
temperatures, which is degraded and cannot act as a regulator; they produce two- to fivefold
more virus m R N A than wild type virus without an increase in genomic R N A synthesis
(Clinton et al. 1978).
In interferon-treated cells infected with VSV under the experimental conditions such as
illustrated in Fig. 5, both cellular and virus protein synthesis are drastically reduced
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Interferon action on R N A and protein synthesis
383
approx. 5 h p.i. This general inhibition of protein synthesis is reflected by the reduced
polysome content of interferon-treated infected cells (Fig. 6) and by the failure of a relatively
large proportion of virus mRNA to associate with polysomes (Table 2).
This inhibition &protein synthesis may be due to the activation of translational inhibitors
in interferon-treated cells. At least two enzymic activities are induced by interferon in a
variety of cells, a 2'5'oligo(A) polymerase and a protein kinase (reviewed by Baglioni,
I979). The 2'5'oligo(A) polymerase synthesizes a series of oligonucleotides (Kerr & Brown,
I978) which activate an endonuclease; the protein kinase phosphorylates and inactivates a
factor necessary for the initiation of protein synthesis (for references, see Baglioni, I979).
Both these enzymes require the presence of double-stranded (ds) RNA and ATP for activity.
A partially dsRNA structure has been isolated from ceils infected by VSV under conditions
whereby virus RNA replication is the predominant synthetic event (Wertz, I978) and
possibly this may activate the enzymic activities induced by interferon,
The VSV mRNA accumulated in interferon-treated cells is of normal size and we have
been unable to detect products of virus mRNA breakdown (unpublished data). This
suggests, though by no means proves, that the endonuclease activated by 2'5'oligo(A) is
not the primary cause of the inhibition of protein synthesis observed late in the infectious
cycle. Perhaps the protein kinase may be involved in this inhibition, since a decreased rate
of initiation would promote polysome run-off and decrease the rate of mRNA entry into
polysomes, the main features of the inhibited protein synthesis observed in interferontreated cells at about 5 h p.i. In a recent investigation, however, Gupta (I979) failed to
detect increased levels of activated protein kinase in interferon-treated cells infected by
VSV. Possibly, under the conditions of these experiments, protein synthesis was not significantly inhibited (no study of the rate of protein synthesis was reported) and the protein
kinase was not activated. Perhaps this activation occurs only when enough dsRNA
structures involved in virus replication have been formed.
Inhibition of protein synthesis was previously observed by Metz & Esteban (I972) in L
ceils treated with interferon and infected with vaccinia virus. The polyribosomes were found
disaggregated in these cells, and Metz et al. (x975) suggested that this resulted from inhibition of the initiation of polypeptide synthesis. The vaccinia RNA synthesized in infected
cells contains a small amount of dsRNA (Colby & Duesberg, I969; Duesberg & Colby,
1969) and this RNA may be responsible for the reduced protein synthesis observed by
Metz & Esteban (2972).
Other explanations for the inhibition of VSV mRNA translation in interferon-treated
cells cannot be excluded at this time. An impairment of mRNA cap methylation has been
reported by Desrosiers & Lengyel (I977) in interferon-treated L cells infected by reovirus.
The virus mRNA synthesized in these cells contains 50% less 'cap I I ' relative to control
cells. Muthukrishnan et al. 0978) have shown, however, that the z'-o-methylation characteristic of c~p II has, at most, a minor effect upon mRNA binding to ribosomes under in
vitro conditions, since some preferential binding of vaccinia mRNAs containing cap II
was observed only at high input mRNA concentrations. Further analysis of the virus
mRNA synthesized in interferon-treated cells infected by VSV is presently in progress in
our laboratory. Such an analysis and further investigations on the activity of initiation
factors isolated from these cells is necessary to pinpoint the cause(s) of the inhibition of
protein synthesis reported here.
This work was supported by Public Health Service Research Grants AI-II887 and
HL-1771o from the National Institute of Health. The authors are grateful to Michael A.
Minks and Timothy W. Nilsen for suggestions and criticism.
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384
M. S I M I L I , F. DE F E R R A A N D C. B A G L I O N I
REFERENCES
BAGLIONI, C. (1979). Interferon induced enzymatic activities and their role in the antiviral state. Cell I7,
255-264.
RANERJEE, A. K. & RHODES, D. P. (1973). In vitro synthesis of RNA that contains polyadenylate by virionassociated RNA polymerases of vesicular stomatitis virus. Proceedings of the National Academy of
Sciences of the United States of America 70, 3566-357OBAXT, B., SONNABEND,J. A. & BABLANIAN,R. (I977). Effects of interferon on vesicular stomatitis virus transcription and translation. Journal of General Virology 35, 325-334.
CLINTON, ~. M., LITTLE, S. P., HAGAN,R. S. & HUANG,A. S. 0978). The matrix (M) protein of vesicular stomatitis
virus regulates transcription. Cell xS, I455-1462.
COLBY, C. & DUESBERG,P. H. 0969). Double-stranded R N A in vaccinia-virus infected cells. Nature, London
222, 94o-944.
DESROSIERS,R. C. & LENGYEL,P. (I 977). Impairment of reovirus m R N A cap methylation in interferon-treated
L cells. Federation Proceedings 36, 8 ~2.
DUESBERG,P. rE & COLaV, ¢. (I969). On the biosynthesis and structure of double-stranded RNA in vacciniavirus infected cells. Proceedings of the National Academy of Sciences of the United States of America
64, 396-4o3.
~UPTA, S. L. 0979). Specific protein phosphorylation in interferon-treated uninfected and virus-infected
mouse L929 cells: enhancement by double-stranded RNA. Journal of Virology 29, 301-31I.
KERR, L M. & BrtOWY, R. ~. 0978). pppA2'p5'A2'p5'A: An inhibitor of protein synthesis synthesized with art
enzyme fraction from interferon-treated cells. Proceedings of the National Academy of Sciences of
America 75, 256-260LAEMMLI,U. K. (I970). Cleavage of structural proteins during the assembly of the head of the bacteriophage
T4. Nature, London227, 68o-685.
LASKEY, R. A. & MILLS, .~. D. (1975). Quantitative film detection of 3H and 14C in polyacrylamide gels by
fluorography. European Journal of Biochemistry 56, 335-34 I.
LODISI~, H. r. (I976). Translational control of protein synthesis. Annual Review of Biochemistry 45, 39-72.
MANDERS, E. K., TILLES, J. G. & HUANG, A. S. (I972). Interferon-mediated inhibition of virion-directed transcription. Virology 49, 573-58 I.
MARCUS,P. L, ENGELHARDT,D. L., HUNG, J. M. & SEKELLICK,M. J. (I97I). Interferon action: inhibition of VSV
RNA synthesis induced by virion-bound polymerase. Science x74, 593-598.
MARCUS,P. L & SEKELLICK,M. J. 0978)- Interferon action. III. The rate of primary transcription of vesicular
stomatitis virus is inhibited by interferon action. Journal of General Virology 38, 39I-4o8.
METZ, D. H. & ESTEBAN,M. (I972). Interferon inhibits viral protein synthesis in L cells infected with vaccinia
virus. Nature, London 238, 385-388.
METZ, D. H., ESTEBAN,M.& DANIELESCU,G. (I975). The effect of interferon on the formation of virus polyribosomes in L-cells infected with vaccinia virus. Journal of General Virology 27, 197-2o9.
MUTHUKRISHNAN, S., MOSS,B., COOPER,J. A. & MAXELL,E. S. (1978). Influence of 5'-terminal structure on the
initiation of translation of vaccinia virus mRNA. Journal of Biological Chemistry 253, I7to-I715.
REPIK, P., FLAMAND,A. & BISHOP,D. H. L. (I974). Effect of interferon upon the primary and secondary transcription of VSV arid influenza viruses. Journal of Virology x4, I I69-It78.
ROSE, J. K. & LODmH, H. r. (I976). Translation in vitro of vesicular stomatitis virus m R N A lacking 5'-terminal
7-methylguanosine. Nature, London 262, 32-37 .
"rHACORE, H. R. (I978). Effect of interferon on transcription and translation of vesicular stomatitis virus in
human and simian ceil cultures. Journal of General Virology 4 x, 421-426.
WEBtR, L. A., r~MAN, E. & BAOLIONI, C. (I975). A cell-free system from HeLa cells active in initiation of
protein synthesis. Biochemistry I4, 5315-532L
VCEBt~t,L. A., SlMILI,J. & BAGLIONI, C. (I979). Binding of viral and cellular messenger RNA's to ribosomes in
eukaryotic cell extracts. Methods in Enzymology 60, 351-36o.
WER:rZ, G. W. 0978). Isolation of possible replicative intermediate structures from vesicular stomatitis
virus-infected cells. Virology 85,271-285.
(Received i o April I979)
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