Download Inhibition of Protein Synthesis by Vaccinia Virus. II. Studies on the

J. gen, Virol. (I979), 44, 625-638
625
Printed in Great Britain
Inhibition of Protein Synthesis by Vaccinia Virus. II. Studies
on the Role of Virus-induced R N A Synthesis
By M. S C H R O M * AND R. B A B L A N I A N t
Department of Microbiology and Immunology, State University of New York,
Downstate Medical Center, Brooklyn, New York 11203, U.S.A.
(Accepted I6 February I979)
SUMMARY
Cytoplasmic RNA synthesis can be detected in vaccinia virus-infected HeLa cells
in the presence of 2/zg/ml but not 2o/*g/ml of actinomycin D. When RNA synthesis
is observed protein synthesis is inhibited in infected, treated cells. We had
previously noted that such a correlation may also be observed in infected, cycloheximide-treated cells. If actinomycin D (20/zg/ml) is added to these cells at various
times after infection and treatment, the inhibition of protein synthesis seen upon
removal of cycloheximide does not continue beyond the point to which it had
developed before the actinomycin D was added. These results indicate that the
inhibition of protein synthesis can be correlated with the amount of cytoplasmic
RNA synthesized in infected cells and that this RNA synthesis and the subsequent
inhibition of protein synthesis can be prevented by sufficiently high concentrations
of actinomycin D. The cytoplasmic RNA which is synthesized does not appear to
consist of double-stranded RNA nor of extensive self complementary regions. The
cytoplasmic RNA synthesized in infected, cycloheximide treated cells appears to
consist of early virus mRNA which can function as mRNA in vitro in a cell-free
system derived from normal cells. An examination of the phosphorylation of
ribosomal proteins shows six additional phosphoproteins in infected cells, two
of which may be observed in infected cycloheximide-treated ceils, suggesting that
phosphorylation of ribosomal proteins cannot be directly correlated with the
inhibition of overall protein synthesis seen in infected cycloheximide-treated cells.
INTRODUCTION
Host protein synthesis is sharply inhibited by vaccinia virus infection (Kit & Dubbs, 1962;
Shatkin, 1963; Holowczak & Joklik, I967; Salzman & Sebring, I967). Two hypotheses have
been advanced to explain this inhibition. The first suggests that the inhibition of protein
synthesis in vaccinia-infected HeLa cells was the result of a component of the virion (Moss,
1968). Infection of cells (25, 50 or I oo p.f.u./cell) in the presence of actinomycin D (5 .?~g/ml)
resulted in multiplicity dependent inhibitions of protein synthesis. Furthermore, cells
infected with 25 p.f.u./cell in the presence of both actinomycin D (5 #g/ml)and cycloheximide (300/zg/ml) failed to resume protein synthesis upon removal of the drug. This,
with the observation that u.v.-inactivated virus also inhibited protein synthesis, led to the
conclusion that a virion component was responsible for the inhibition (Moss, I968). The
* Present address: Department of Microbiology and Immunology, Albany Medical College, Albany,
N.Y. I2208, U.S.A.
t Author to whom reprint requests should be made.
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M. S C H R O M A N D R. B A B L A N I A N
second hypothesis states that the inhibition of protein synthesis can be correlated with
virus-induced RNA synthesis (Babtanian, I975; Bablanian et al. I978). It was found
that while vaccinia-infected (300 particles/cell), cycloheximide-treated (300/zg/ml), L and
HeLa cells failed to resume protein synthesis upon removal of the drug, similarly infected
LLC-MK2 cells did resume protein synthesis upon cycloheximide reversal. We have prepared
cell-free amino acid incorporating systems from such infected cells in which the inhibition of
protein synthesis observed in vivo may be reproduced in vitro (Schrom & Bablanian, I979).
It was also determined that the onset and extent of inhibition of protein synthesis in
vaccinia-infected cells correlates well with the rate of cytoplasmic virus RNA synthesis in
infected cells. HeLa and L cells show an increasing rate of cytoplasmic RNA synthesis
while in LLC-MK2 cells the rate of RNA synthesis is lower and does not increase with time.
The host dependent difference emphasizes the importance of the cell in a virus-cell interaction (Bablanian, 1975; Bablanian et al. I978).
The use of inhibitors of RNA synthesis has led to conflicting conclusions on the requirement for RNA synthesis in the establishment of inhibition of protein synthesis. Moss (I968)
found that 5/zg/ml of actinomycin D could not prevent the inhibition of protein synthesis.
Previous work in our laboratory showed that 20/zg/ml of actinomycin D could prevent the
inhibition of protein synthesis (Bablanian et al. 1978). We did, however, in agreement with
the observations of Moss (I968), find that lower concentrations of actinomycin D (t/zg/ml)
allowed the development of inhibition of protein synthesis. Previous work has demonstrated that, in the presence of I #g/ml of actinomycin D, early virus RNA synthesis occurs
in infected L cells (Metz & Esteban, t972). Furthermore, Rosemond-Hornbeak & Moss
(I975) demonstrated that a small poly(A)-rich RNA is made and thus may contribute to
the virus-induced inhibition of host polypeptide synthesis. In order to clarify this situation
we have examined RNA synthesis in vaccinia-infected cells, treated with various concentrations of actinomycin D. Our results suggest that higher concentrations of actinomycin D
may be necessary to penetrate the virus cores in order to effectively inhibit DNA-dependent
RNA synthesis. Our previous work (Bablanian, I975; Bablanian et al. I978) has indicated
that, in cells infected with vaccinia virus in the presence of cyclohenimide, the rate of
cytoplasmic RNA synthesis correlates well with the inhibition of protein synthesis. We
have further examined this phenomenon and will present evidence to show that the inhibition is also related to the amount of RNA synthesized.
It is not known what kind of cytoplasmic RNA is responsible for the inhibition nor how
it acts. There is ample evidence that dsRNA can inhibit protein synthesis (Hunt & Ehrenfeld,
I97t; Robertson & Mathews, I973; Hunter et al. 1975; Lebleu et al. I976; Roberts et al.
I976). It is also known that such a molecule is synthesized in vaccinia-infected cells (Colby
& Duesberg, I969; Duesberg & Colby, I969; Colby et al. 1970. However, it is not known
whether the synthesis of such a molecule can be correlated with the inhibition of protein
synthesis. Evidence will be presented in this communication which indicates that dsRNA
is not synthesized in substantial amounts under conditions where inhibition of protein
synthesis may be observed. The inhibition of protein synthesis by dsRNA has been shown
to be mediated by phosphorylation of initiation factor IF-3 (eIF-2) (Kaempfer & Kaufman,
I973). We have examined the phosphorylation of ribosomal proteins in vaccinia-infected
cyclo-heximide-treated cells and will present evidence which suggests that such a process is
not involved in the inhibition of overall protein synthesis.
METHODS
Virus and cell cultures. The growth of vaccinia virus (strain WR) and the maintenance of
HeLa and L-929 monolayers was as described previously (Bablanian et al. 1978).
Measurement o f protein
synthesis from
in vivo.
Protein synthesis in infected
cells was measured
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as described previously (Bablanian et al. I978). Protein determinations are carried out
according to Bramhall et al. (I969) and hot trichloroacetic acid (TCA)-insoluble incorporation is measured according to Mans & Novelli (~960.
Measurement of RNA synthesis in vivo. The rate of RNA synthesis was determined by
the method of Becker & Joklik (~964), a procedure which assumes that most of the RNA
labelled in the cytoplasm of infected cells during a IO rain pulse with 3H-uridine is from the
virus.
Polyacrylamide gel electrophoresis (PAGE). 35S-methionine-IabelIed proteins were analysed by PAGE in a vertical gel slab apparatus. Resolving gels ~5o mm long and I'5 mm
thick and containing gradients of acrylamide monomer of 5 to I5% were cast using the
buffer system of Laemmli 097o). Electrophoresis was at r2o V until the tracking dye
reached the bottom of the gel. The gel was washed in 7 % acetic acid for several hours and
dried under vacuum and autoradiographed (Fairbanks et al. I965) using DuPont Cronex
z X-ray film.
Measurements of double-stranded RNA in infected cells. Cells were infected or mockinfected in the presence of 2o #Ci/ml of 3H-uridine. At 3"5 h p.i. cytoplasmic RNA was
extracted by a modification of the method of Oda & Joklik (t967). Cells were washed with
cold RSB (IO mM-tris-HCl, pH 7"6; IO mM-KCI; I'5 mM-MgC12), scraped into z ml of
RSB and disrupted by 2o strokes of a Dounce homogenizer. The homogenates were centrifuged at 2ooo rev/min for IO rain and I o % SDS was added to the supernatant to o'5%.
After 3 to 5 rain at 37 °C, sodium acetate (pH 5.o) was added to o.I M. Sodium perchlorate
and ethylene diamine tetra-acetic acid (EDTA) were added to I M and 1.5 m u respectively.
An equal vol. of chloroform:isoamyl alcohol (24:1) was added and, after mixing, the
samples were centrifuged to separate the phases. The aqueous layer was re-extracted one to
five times until the interface between the phases remained clear. The RNA was then precipitated at - 2o °C overnight by addition of z vol. of ethanol. The RNA was collected by
centrifugation and dissolved in 2OO/zl of TNE buffer Oo mra-tris-HC1 pH 7"6, o-i M-NaC1,
I mM-EDTA). Portions (IO #1) of this RNA were precipitated with I ml of 7"5 % TCA
after addition of too #g carrier yeast RNA. For ribonuclease treatment Ioo/zl samples were
diluted to o'5 ml in T N E and 2o #g/ml of bovine pancreatic ribonuclease (Worthington)
dissolved in 4 x SSC (I5O mM-NaC1, I5 m i - s o d i u m citrate, I rnM-EDTA-SSC) was added.
After 30 min at 37 °C, IOO/zg of carrier RNA and 2 ml of 7"5 % TCA was added. The precipitates were collected on Whatman G F / C filters after 3o min at 4 °C, dried and counted.
Self annealing was performed by bringing the samples to the concentration of 4 × SSC and
incubating at 65 °C for I8 h. A sample was removed to measure total acid-precipitable
radioactivity and the balance was treated with ribonuclease as described. The counts were
corrected to a constant vol. of IOO #1.
Examination of phosphorylation of ribosomal proteins in infected cells. L-929 cells were
infected (3oo particles/cell) or mock-infected in the presence or absence of 30o/zg/ml of
cycloheximide. The infection was carried out in reinforced Eagles' medium (Bablanian et aL
I965) from which phosphate had been omitted and in the presence of 2 % dialysed foetal
bovine serum. The cells were labelled from the start of the infection with 5o #Ci/ml of
32PO 4 (carrier-free, The Radiochemical Centre, Amersham, Bucks, U.K.). At 3"5 h p.i. the
cells were washed with cold 30 mM-tris-HCl, pH 7"4; I4o mi-NaC1 and then with hypotonic
medium (IO mM-tris-HCl, pH 7"4, IO mM-KCI, 1.5 mM-magnesium acetate, 7 mM-2mercaptoethanol). The cells were drained nearly dry and scraped into what remained of the
buffer. After 5 min the cells were homogenized by 20 strokes of a glass Dounce homogenizer.
The homogenate was brought to 30 mM-tris-HC1 (pH 7.4), 90 mM-KC1, 3"5 raM-magnesium
acetate and 7 mM-2-mercaptoethanol and centrifuged at IOOOOg for Io min. Half of the
resulting supernatant was made 0"5 M-KCI by dropwise addition of 4 M-KCI. Both the KCI
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preparations
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centrifuged for 60 minbyat 50000 rev/min in the
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M. S C H R O M A N D R. B A B L A N I A N
Beckman type 65 rotor. The resulting pellets were subjected to PAGE and autoradiography
as described above.
Preparation of cell-free amino acid-incorporating system. Cell-free amino acid-incorporating
systems were prepared from L cells grown in suspension. The cells were washed three times
with cold tris-saline (3o mM-tris-HC1, pH 7"4, I4o mM-NaCI) and suspended in I'5 packed
cell vol. of hypotonic medium (lo mM-tris-HCl, pH 7"4, IO mM-KCI, I'5 mM-magnesium
acetate, 7 mM-2-mercaptoethanol). After 5 min the cells were homogenized by 20 strokes of
a glass Dounce homogenizer. The homogenate was brought to 30 mM-tris-HCl (pH 7"4),
9o mM-KCI, 3"5 mM-magnesium acetate and 7 mM-2-mercaptoethanol and centrifuged at
10000 g for ~o min.
The supernatants were pre-incubated at 37 °C for 40 min after addition of adenosine
triphosphate (ATP, I mM), guanosine triphosphate (GTP; o.1 mM), cytidine triphosphate
(CTP, o.6 raM), creatine phosphate (to ms~), creatirte kinase (o. 16 mg/ml) and 40 mM each
of all of the amino acids (all obtained from Calbiochem, La Jolla, Calif., U.S.A.). The
extract was chilled and desalted by passage through Sephadex G25 columns prior to storage
and assay. Sephadex columns were prepared by equilibration of the gel in 30 mM-tris-HC1
(pH 7"4), lOO rnM-KCI, 5 raM-magnesium acetate, 7 mi+mercaptoethanol and 5 % (v/v)
glycerine. The extract ($io) was passed through the column and the fractions containing the
greatest amount of protein (monitored visually by turbidity), corresponding to the void vol.,
were pooled and stored at - 7 0 °C in IOO#1 samples.
Assay of cell-free protein synthesis directed by RNA extracted from infected cells. The
RNA extracted from infected cells as described above was added at 3 #g per assay to cellfree protein synthesizing systems prepared as described above. The reaction mixture
contained, in a total vol. of 50 #1, tris-HC1 (pH 7"4), 35 raM; 2-mercaptoethanol, 9 m i ;
ATP, I mM; GTP, o.1 mM; CTP, 0.6mM; creatine phosphate, 1OmM; creatine kinase,
o.16 mg/ml; 85S-methionine, IOO~tCi/ml; 40/ZM each of the other 19 amino acids; lO #1 of
the appropriate Sio; 50 mM-KCI and 3"5 mM-magnesium acetate. For endogenous mRNA
in non-pre-incubated extracts KCI was usually 1IO mM and magnesium acetate 4"5 mM,
unless otherwise indicated. L cell mRNA and globin mRNA were translated at 5° mM-KC1
and 3"5 mM-magnesium acetate. The reaction was carried out at 3° °C for I2O min. Ten #1
samples were removed and placed on filter paper discs (Whatman no. 42, 2"54 cm) which
had previously been spotted with 1% (w/v) casamino acids. When all of the samples had
been collected the papers were dried and hot TCA-insolubIe radioactivity was measured as
described above. The remaining portion of the reaction mixture was mixed with an equal
vol. of electrophoresis sample buffer for polyacrylamide gel electrophoresis and autoradiography.
RESULTS
RNA synthesis in vaccinia-infected cells in the presence of actinomycin D
Previous studies using actinomicin D to inhibit vaccinia virus RNA synthesis (Moss,
1968; Bablanian, 1975; Rosemond-Hornbeak & Moss, 1975; Bablanian et aL 1978) have
left unresolved the question of whether RNA synthesis is required for establishment of
inhibition of protein synthesis in vaccinia-infected cells. In order to clarify this question
HeLa cells were infected with 9o0 particles per cell in the presence of 2 or 2o #g/ml of
actinomycin D. At various times after infection cytoplasmic RNA synthesis and protein
synthesis were measured. Fig. I shows that RNA synthesis as measured by uridine incorporation, can be detected in the presence of 2 #g/ml but not 2o/tg/ml actinomycin D. When
protein synthesis is examined 3"5 h p.i. (Fig. 2) it can be seen that 2/zg/mI actinomycin D
is not sufficient to prevent the virus-induced inhibition of protein synthesis. However, when
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RNA and inhibition of protein by vaccinia
i
,
629
,
14
12
10
'7
0
8
X
E
O r
0
i
I
I
60
120
180
Time (min)
Fig. i. Cytoplasmic RNA synthesis in vaccinia virus-infected cells in the presence of different
concentrations of actinomycin D. HeLa cells were infected with 900 particles/cell of vaccinia virus
in the presence of 20 #g/ml or 2 #g/ml actinomycin D or in the presence of 300 #g/ml cycloheximide. At the indicated times p.i. the ceils were pulse-labelled with 3H-uridine and cytoplasmic
RNA synthesis was measured as described in the Methods. The background incorporation in
treated uninfected cells has been subtracted. 0 - - 0 Cells infected in the presence of 2o #g/ml
actinomycin D; A--A, cells infected in the presence of 2 #g/ml actinomycin D; I I - - I ; cells
infected in the presence of cycloheximide.
cells are infected in the presence of 20/~g/ml actinomycin D the inhibition of protein
synthesis is largely prevented. From this we conclude that when sufficient concentrations of
actinomycin D are present, the synthesis of virus-induced R N A is prevented and protein
synthesis is not inhibited.
It should be noted that, in agreement with the results of others (Rosemond-Hornbeak &
Moss, r975), we have noted incorporation of 3H-adenosine even in the presence of 20/zg/ml
of actinomycin D which is consistent with the observation that polyadenylation of vaccinia
m R N A is not inhibited by actinomycin D (Kates & Beeson, x97o).
Relationship between amount of cytoplasmic RNA synthesized in infected cells and
inhibition of protein synthesis
Our previous results (Bablanian et aL I978) have shown that when HeLa cells are infected
with vaccinia virus in the presence of cycloheximide the rate of R N A synthesis increases
with time and this increase in the rate is related to the inhibition of protein synthesis. It is
possible that this inhibition is related to the amount of R N A present or to an effect which
develops with time due to the presence of a specific class of early RNA. In order to distinguish these possibilities HeLa cells were infected in the presence of cycloheximide and,
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M. S C H R O M A N D R. B A B L A N I A N
2
3
4
5
6
Fig. 2. Effect of actinomycin D and cycloheximide on virus and host polypeptide synthesis in
vaccinia virus-infected HeLa cells. Cells were infected with 90o particles/cell or infected-treated in
the presence of actinomycin D (ACD) or cycloheximide. At 3 h p.i. the cells were washed free of
cycloheximide and all cultures were labelled for 30 rain with 8sS-methionine. PAGE and autoradiography were performed as described in the Methods. i, Virus + 20/zg/ml ACD; 2, 2o #g/ml ACD;
3 , virus+2/~g/ml A C D ; 4, 2/zg/ml ACD; 5, virus+30o/zg/m I cycloheximide; 6, 3oo/~g/ml
¢ycloheximide; 7, virus; 8, cells.
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RNA and #thibition o f protein by vaccinia
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J
lOO
80
60
40
20
0
i
0
60
I
120
Time (rain)
I
180
I
240
Fig. 3. Relationship between amount of cytoplasmic RNA synthesized in infected cells and
inhibition of protein synthesis. HeLa cells were infected (goo particles/cell) in the presence of
300/zg/ml cycloheximide. Replicate cultures were treated with 20/tg/ml actinomycin D at 60, I2O
or I8o min p.i. At the indicated times p.i., the cells were washed free of cycloheximide, labelled
with s~S-methionine for 30 min and sp. act. was determined as described in the Methods. Results
are expressed as percentage of uninfected control. Q--Q, No actinomycin D; actinomycin D added
at: I I - - I I , 6o min; A--A, zzo min; T - - T , 18o min.
at various times p.i., 20/zg/ml actinomycin D was added to stop further R N A synthesis. The
cycloheximide was removed from treated cultures and protein synthesis was measured as
described in the Methods.
Fig. 3 shows that when R N A synthesis is interrupted by the addition of actinomycin D
the inhibition of protein synthesis does not progress beyond the point to which it had been
reduced before the addition of the drug. This suggests that the inhibition of protein synthesis
is related to the amount of R N A synthesized and not to a catalytic effect produced by an
early R N A species. What remains unclear is what R N A is responsible for this inhibition.
Relationship of dsRNA to inhibition of protein synthesis in infected cells
The one R N A species which has been shown to be an inhibitor of protein synthesis is
double-stranded R N A (Hunt & Ehrenfeld, I 9 7 I ; Robertson & Mathews, I973; Hunter
et al. I975; Lebleu et al. I976; Roberts et al. t976). This molecule has also been shown to
be synthesized in vaccinia-infected cells (Colby & Duesberg, I969; Duesberg & Colby, 1969;
Colby et al. t97i), however, it has not been shown that the synthesis o f d s R N A may be
specifically correlated with the inhibition of protein synthesis in infected cells. L-929 cells
were infected or mock-infected in the presence or absence of cycloheximide and in the
presence of 8H-uridine. At 3"5 h p.i. total cytoplasmic R N A was extracted and the total and
ribonuclease-resistant radioactivity was measured. It has previously been shown (Kates &
McAuslan, I967) that early virus R N A synthesis proceeds at an accelerated rate in the
presence of cycloheximide. Table i shows that while we also observe a large increase in the
amount of acid-precipitable radioactivity found in infected treated cells, the percentage of
this which is ribonuclease resistant does not increase in infected treated cells. In the absence
of cycloheximide, infected cells show an increase in the percentage of nuclease-resistant
material. The infected cycloheximide-treated cells show an increase in the absolute amount
of ribonuclease-resistant RNA. Although it is possible that this does represent the
synthesis of small amounts of dsRNA, and despite the fact that nuclease resistance is not
an absolute criterion for double-strandedness, we suggest that the failure of this material
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Table t. Cytoplasmic RNA* resistant to ribonuclease (RNase) from vaccinia-infected
L- 929 cells
Cells
RNA
~
~
Cycloheximide
Total
~H-uridine
(ct/min)
RNaseresistant
(ct/min)
%
resistant
Infected
1571o
387
2.46
Infected
+
9935o
773
o'78
Uninfected
3716o
239
o'64
Uninfected
+
34 o3o
257
o'76
* Cells were infected or mock-infected in the presence or absence of 300 #g/ml cycloheximide and in the
presence of 2o #Ci/ml aH-uridine. After 3'5 h total cytoplasmic RNA was extracted and treated with ribonuclease as described in the Methods.
Table 2. Self annealing* of cytoplasmic RNA from vaccinia-infected L-929 cells
RNA
r
-Cells
Total
~ 3H-uridine
Cycloheximide
(ct/min)
A
RNaseresistant
(ct/min)
Infected
15 205
Infected
+
98 o32
Uninfected
37084
Uninfected
+
32 581
* Total cytoplasmic RNA, prepared as described in Table I, was
65 °C before RNase treatment.
%
resistant
295
1.94
1392
1.4 I
472
t" 27
456
1.4o
self-annealed in 4 x SSC for 18 h at
to produce an increase in the percentage o f nuclease-resistant R N A makes the synthesis
o f large amounts o f d s R N A unlikely.
It is possible that there m a y exist in infected cells complementary R N A sequences which
have not formed double-stranded molecules. In order to exclude this possibility the extracted
R N A was self annealed for I8 h at 65 °C in 4 x SSC and again subjected to ribonuclease
digestion. Again the only sample which shows an increased nuclease resistance is the infected untreated cells (Table 2). This is in agreement with the observations o f Boone & Moss
0 9 7 8 ) that early vaccinia R N A does not contain extensive self complementary sequences.
These results suggest that the extensive inhibition o f protein synthesis we observe under
these conditions cannot be correlated with the synthesis o f large a m o u n t s o f doublestranded R N A . However, in view o f the approximate doubling o f the absolute a m o u n t o f
nuclease-resistant R N A in the infected-treated cells, the involvement o f d s R N A cannot be
definitely excluded.
It is possible that small a m o u n t s o f d s R N A functioning in a catalytic fashion (Hunter
et al. I975) might act to inhibit protein synthesis. One situation in which this has been shown
to occur is t h r o u g h the phosphorylation o f IF-3 (eIF-2) (Kaempfer & K a u f m a n , I973). It is
k n o w n that d s R N A stimulates phorphorylation o f ribosomal proteins in interferon-treated
cells (Roberts et al. I976) and also that ribosomal proteins are phosphorylated in vacciniainfected cells (Kaerlein & Horak, I976 ). It was therefore o f interest to examine the phosphorylation o f ribosome-associated proteins in infected treated cells.
Role of protein phosphorylation in the inhibition of protein synthesis
L-929 ceils were infected or mock-infected in the presence or absence o f cycloheximide
and in the presence o f 5o/zCi/ml 3~P-H3PO a. At 3"5 h p.i. crude unwashed ribosomes and
KC1 washed ribosomes were prepared as described in the Methods and analysed by P A G E .
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1
2
3
4
5
6
7
C
D
!~~ ~iii~i~i~iii~/i/!~ili?/i!i~iiil¸~ i
i!i
/ ! :/ i ~I ~
Fig. 4. Phosphorylation of ribosomal proteins in vaccinia-infected cells. L-9z9 cells were infected
or mock-infected (300 particles/cell) in the presence or absence of 300 #g/ml cycloheximide and in
the presence of 50 #Ci/ml 32PO~ in phosphate-free REM. At 3"5 h p.i. unwashed and KC1 washed
ribosomes were prepared as described in the Methods and subjected to P A G E a n d autoradiography.
I, unwashed infected ribosomes; 2, unwashed infected-cycloheximide treated ribosomes; 3, unwashed uninfected ribosomes; 4, unwashed uninfected-cycloheximide treated ribosomes; 5, KC1
washed infected ribosomes; 6, KCI washed infected-treated ribosomes; 7, KCI washed uninfected
ribosomes; 8, KCI washed uninfected-cycloheximide treated ribosomes. The additional phosphorylated bands are indicated at their left and identified by letters. The approx, mol. wt. range was
from 45000 to 10000.
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M. S C H R O M A N D R. B A B L A N I A N
Table 3. Addition of total cytoplasmic RNA from vaccinia-infected L-929 cells
to cell-free protein-synthesizing systems*
C"ells from which RNA was extracted
None
Infected
Infected-cycloheximide treated
Uninfected
Uninfected-cycloheximide treated
35S-methionine
(ct/min)
1985~
t 4445
72454
22 653
35 154
* Pre-incubated cell-free systems were prepared from normal cells as described in the Methods. R N A
was added at 3 #g/5o #1 assay.
It can be seen (Fig. 4) that there are six phosphorylated proteins associated with the unwashed ribosomes of the infected untreated cells not found in infected cells. Five of these
(Bands, A, C, D, E, F) remain after KCI washing. The infected treated cells contain two of
these proteins. The three major phosphorylated bands (Bands B, C, E) in the infected
untreated cells are absent in the cycloheximide-treated infected cells. Since the infected
treated cells manifest a complete inhibition of protein synthesis in the absence of extensive
phosphorylation, we suggest that phosphorylation of ribosome-associated proteins is not
involved in the inhibition of protein synthesis. It may be that the two phosphoproteins
common to the infected and infected treated cells (Bands A, F) are related to the selective
inhibition of cellular protein synthesis but it does not appear that the overall inhibition of
protein synthesis can be correlated with an increase in protein phosphorylation in infected
cycloheximide-treated cells.
Addition of RNA from infected treated cells to cell-free amino acid-incorporating systems
In order to determine whether the increase in cytoplasmic RNA represented messenger
RNA, the total cytoplasmic RNA extracted from infected, infected treated, uninfected
treated and uninfected cells was added to cell-free amino acid-incorporating systems prepared from normal cells. When RNA from infected treated cells is added to pre-incubated
cell-free systems it is capable of directing amino acid incorporation in vitro (Table 3) even
though mRNA has not been specifically extracted. A slight stimulation is observed with
RNA from uninfected treated cells. However, when the cytoplasmic RNAs from infected
untreated or uninfected untreated cells were added to cell-free systems, no such stimulation
of incorporation was observed. When the products of these reactions were analysed by
PAGE (Fig. 5) several bands were seen which do not correspond to the endogenous products. We presume, therefore, that these bands represent virus proteins, the translation of
which was directed by virus mRNAs which had accumulated in the presence of cycloheximide.
DISCUSSION
The results presented in this communication support the idea that RNA synthesis is
required for the inhibition of protein synthesis by vaccinia virus. We have confirmed the
results of Shatkin (i963) and Moss (1968) that cellular protein synthesis is inhibited in the
presence of actinomycin D at concentrations up to 5 #g/ml. However, we have also shown,
as noted by Metz & Esteban (t972) and Rosemond-Hornbeak & Moss (t975), that RNA
synthesis can be observed in cells infected in the presence of as much as 5/~g/ml actinomycin
D. When sufficient concentrations of the drug are added to completely prevent RNA synthesis (Fig. I), inhibition of protein synthesis is not observed (Fig. z). While zo/zg/ml
actinomycin D may appear excessive, it may be that lower concentrations of the drug fail
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RNA and inhibition of protein by vaccinia
1
2
3
4
635
5
Fig. 5. Protein synthesis in vitro directed by RNA from infected cells. L-929 cells were infected or
mock-infected (300 particles/cell) in the presence or absence of 300 #g/ml of cycloheximide. At
3"5 h p.i. total cytoplasmic RNA was extracted and added to pre-incubated cell-free proteinsynthesizing systems as described in the Methods. I, no RNA added; 2, RNA from infected cells;
3, RNA from infected cycloheximide-treated cells; 4, RNA from uninfected cells; 5, RNA from
uninfected cycloheximide-treated cells.
to p e n e t r a t e the virus core structure as suggested b y M e t z & E s t e b a n (I972). The lower
c o n c e n t r a t i o n s m a y be sufficient to interfere with R N A synthesis so that the p r o d u c t is
a b e r r a n t a n d n o n - t r a n s l a t a b l e . This w o u l d be consistent with the o b s e r v a t i o n s o f R o s e m o n d H o r n b e a k & M o s s (I975) t h a t small a b e r r a n t p o l y ( A ) - r i c h R N A molecules are synthesized
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636
M. SCHROM AND R. BABLANIAN
in the presence of 5 #g/ml actinomycin D. This sort of molecule may be responsible for the
inhibition of protein synthesis in the presence of actinomycin D. However, inhibition of
protein synthesis also occurs in the absence of actinomycin D (i.e. in the presence of cycloheximide) therefore other mechanisms of inhibition should be postulated.
We have previously observed a relationship between RNA synthesized in the presence of
cycloheximide and inhibition of protein synthesis upon removal of the drug (Bablanian et al.
I978). In the present study we have further examined this relationship. The first approach
we took was to determine whether the inhibition is the result of the catalytic action of some
RNA species or of the accumulation of an inhibitor which acts in stoichiometric amounts.
The results presented in Fig. 3 suggest that the accumulation of RNA is responsible fonthe
development of inhibition of protein synthesis. This accumulation may be interrupted by
addition of zo/zg/ml actinomycin D, and once RNA synthesis is interrupted, the inhibition
does not advance beyond the point to which it had developed when the actinomycin D was
added.
The only situation in which an RNA molecule has been shown to be an inhibitor of
protein synthesis is the case of dsRNA (Hunt & Ehrenfeld, I97r ; Robertson & Mathews,
I973; Hunter et al. 1975; Lebleu et aL 1976; Roberts et al. I976). This inhibition of protein
synthesis has been shown to be the result of inactivation of an initiation factor (Kaempfer &
Kaufman, 1973) which, in reticulocytes and interferon-treated cells, is mediated by phosphorylation (Lebleu et al. I976; Roberts et al. I976; Farrell et al. 1977). Although Kaerlein
& Horak (I976) have shown, as we have also observed (Fig. 4), that ribosomal proteins are
phosphorylated in vaccinia virus-infected cells, our evidence (Table I, Table 2, Fig. 4) does
not suggest the direct involvement of dsRNA or any phosphorylation of ribosomal proteins
in the inhibition of protein synthesis by vaccinia virus in infected-cycloheximide treated
cells.
The results in Table 3 suggest that the greatest proportion of the RNA synthesized in
infected-cycloheximidetreated cells represents early virus messenger RNA and it constitutes
a large enough fraction of the total RNA content to direct protein synthesis in vitro without
oligo(dT) chromatography. It is possible that the accumulation of early mRNA in vast
excess may itself interfere with protein synthesis by some as yet unknown process. We have
recently presented evidence (Schrom & Bablanian, I979) which indicates that the inhibition
of protein synthesis takes place at the level of initiation of new polypeptide chains, an
observation consistent with those of Person & Beaud (I978) and Ben-Hamida & Beaud
(I978) obtained by different methods.
We must therefore postulate that early virus mRNA specifically interferes with initiation
in infected-cycloheximidetreated cells. What cannot be excluded is the possibility that some
aberrant RNA, such as, but not necessarily identical to, the small poly (A)-rich molecules
observed by Rosemond-Hornbeak & Moss (I975) may be synthesized. However, with our
present data, it remains true that the inhibition of protein synthesis in infected treated cells
can best be correlated with the synthesis of early virus mRNA.
The authors wish to thank Santo Scribani for technical assistance. This work is based on
part of the dissertation submitted by M.S. to the School of Graduate Studies of the State
University of New York in partial fulfilment of the requirements for the degree of Doctor of
Philosophy.
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637
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