Download The Regulation of Translation in Reovirus-infected Cells

J. gen. Virol. (1985), 66, 2161-2170. Printedin Great Britain
2161
Key words: reovirus/translation/double-inJection
The Regulation of Translation in Reovirus-infected Cells
By A L B E R T O M U N O Z , M I G U E L A N G E L A L O N S O AND
LUIS CARRASCO*
Departamento de Microbiologia, Centro de Biologia Molecular, Universidad Aut6noma de
Madrid and CSIC, Canto Blanco, Madrid-28049, Spain
(Accepted 21 June 1985)
SUMMARY
The regulation of translation in reovirus-infected cells has been investigated by
double-infection experiments. Different cell lines were able to translate uncapped
encephalomyocarditis (EMC) virus mRNA and capped vesicular stomatitis virus
(VSV) mRNAs both early and late during reovirus infection. These results are not fully
in agreement with a previously suggested model in which, in the early phase of reovirus
infection, only capped mRNAs are translated, whereas in the late phase the cells are
modified to translate exclusively uncapped mRNAs. The observations that EMC virus
and VSV shut down host protein synthesis more efficiently than reovirus translation in
the late phase in double-infections indicate that competition for a messagediscriminatory factor may not be involved in the shut-off of host protein synthesis in
these animal virus-infected cells.
INTRODUCTION
The regulation of translation in virus-infected animal cells has been a matter of intensive
research during the last few years. In most animal systems studied, as for example picornavirusinfected cells, there is a potent inhibition of host protein synthesis after viral infection, followed
by massive translation of viral mRNAs (Carrasco & Smith, 1976, 1980; Lucas-Lenard, 1979). In
other instances, such as in some reovirus-infected cells, host and viral protein synthesis co-exist,
with no apparent shut-down of host translation (Walden et al., 1981 ; Detjen et al., 1982). In
encephalomyocarditis (EMC) virus-infected cells, inhibition of protein synthesis appears to be
related to a modification of membrane permeability which leads to changes in the ionic
conditions in the cells, with the result that translation of cellular mRNA is blocked, while viral
protein synthesis is still favoured (Carrasco & Smith, 1976; Carrasco, 1977; Lacal & Carrasco,
1982). The suggestion that EMC virus mRNA is preferentially translated in infected cells
because it is better able to compete for a limiting factor involved in protein synthesis has also
been proposed (Golini et al., 1976). However, the possibility that competition is the cause of the
shut-off phenomenon in EMC virus-infected cells has been ruled out for two main reasons: (i)
shut-off still occurs even in the absence of viral mRNA production and (ii) if host mRNAs a r e
replaced by more efficient viral mRNAs, then no inhibition of total protein synthesis should be
observed (Steiner-Pryor & Cooper, 1973; Bablanian, 1972; Holland, 1964; Collins & Roberts,
1972; Carrasco & Smitfa, 1980; Mufioz & Carrasco, 1981).
Competition has also been considered to operate in reovirus-infected SC1 cells. Indeed, in a
series of publications, Thach and co-workers have developed a kinetic model to explain the
behaviour of protein synthesis in reovirus-infected cells (Walden et al., 1981; Brendler et al.,
1981 a, b; Godefroy-Colburn & Thach, 1981 ; Detjen et al., 1982). According to this model, viral
and cellular mRNAs compete for a message-discriminatory component that participates in
translation, before the mRNA binds to the 40S initiation complex. This component has been
tentatively identified as initiation factor elF-4A (Ray et al., 1983). Evidence was obtained
showing that reovirus mRNAs are poor initiators relative to host mRNAs. Thus, the following
order in competition was established: EMC virus > host > reovirus mRNA. Therefore, it was
proposed that the translation strategy adopted by reovirus is to overwhelm the cellular synthetic
0000-6557©1985 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
2162
M. MUI~IOZ, M. A. ALONSO AND L. CARRASCO
a p p a r a t u s with a large excess of weakly initiating messages ( W a l d e n et al., 1981). In addition, it
was o b s e r v e d that the degree of c o m p e t i t i o n b e t w e e n host and viral m R N A largely d e p e n d s on
the ionic c o n c e n t r a t i o n in the cell-free system (Brendler et al., 1981a, b).
C o n t r a r y to this view, Skup & M i l l w a r d (1980) h a v e suggested that the i n h i b i t i o n o f
translation after reovirus infection o f L cells occurs by a m e c h a n i s m similar to t h a t already
suggested for poliovirus. The m o d e l proposes t h a t the reovirus m R N A synthesized early in
infection is capped, and the infected cells translate this k i n d o f m R N A efficiently, but not
u n c a p p e d m R N A s . Late in infection, the viral m R N A p r o d u c e d is u n c a p p e d and it is efficiently
translated because the protein-synthesizing m a c h i n e r y has b e e n modified in such a w a y t h a t
only u n c a p p e d m R N A s are r e c o g n i z e d (Skup & Millward, 1980; Skup et al., 1981). H o w e v e r ,
the validity of the reovirus and poliovirus models has b e e n q u e s t i o n e d recently (Detjen et al.,
1982; A l o n s o & Carrasco, 1982b).
T o investigate these models and also to m e a s u r e to w h a t extent c o m p e t i t i o n plays a part in the
regulation o f translation in reovirus-infected cells, we h a v e analysed p r o t e i n synthesis in
reovirus-infected cells, s u p e r i n f e c t e d w i t h different a n i m a l viruses w i t h c a p p e d or u n c a p p e d
m R N A s . C o n t r a r y to Skup and co-workers (Skup & Millward, 1980; Skup et al., 1981) and in
a g r e e m e n t w i t h D e t j e n et al. (1982), the results o b t a i n e d led us to consider t h a t r e o v i r u s - i n f e c t e d
H e L a cells are able to translate c a p p e d viral m R N A s late during virus infection. H o w e v e r , in
reovirus-infected H e L a cells, no i n h i b i t i o n o f cellular protein synthesis is a p p a r e n t at the t i m e
w h e n m o s t viral proteins are synthesized, w h i c h suggests that there is an excess o f translation
c o m p o n e n t s and that no c o m p e t i t i o n b e t w e e n cellular and viral m R N A s is established in intact
infected cells. M o r e o v e r , superinfection o f these cells w i t h E M C virus shuts d o w n cellular
protein synthesis m o r e efficiently t h a n reovirus translation. T h i s is a n o t h e r a r g u m e n t against
c o m p e t i t i o n playing a part in the shut-off p h e n o m e n o n , since, as suggested by W a l d e n et al.
(1981), host m R N A s are m o r e efficient c o m p e t i t o r s t h a n reovirus m R N A s .
METHODS
Cells and viruses. HeLa cells and BHK-21 cells were grown in Dulbecco's modified Eagle's medium (E4D)
supplemented with l0 % calf serum (E4D10 medium) and incubated at 37 °C in a 5 ~ CO2 atmosphere. Poliovirus
type 1 was grown on HeLa cells in E4D medium supplemented with 2 ~ newborn calf serum (E4D2). Vesicular
stomatitis virus (VSV) was grown in BHK-21 cells in the same medium. EMC virus was grown on L929 cells in a
mixture of Eagle's medium, phosphate-buffered saline (PBS) and E4D medium (80 : 15 : 5) supplemented with 1
newborn calf serum.
In all cases, the fraction obtained after removal of cell debris by low-speed centrifugation was used as the source
of virus. Reovirus type 3 Dearing strain was purchased from the American Type Culture Collection.
Virus infection, measurement and analysis of protein synthesis. Cells grown on 24-well plates (Falcon Plastics)
containing 1 ml E4D10 were infected with the indicated virus at the m.o.i, described in each experiment. After 1 h
incubation at 37 °C, the medium was removed and 1 ml E4D2 was added. Time of virus addition was considered as
1 h, and 0 time was taken as the time when the virus was removed. Incubation at 37 °C was continued until the
labelling period. For this purpose, 0.5 ml methionine-free E4Dl medium and 5.4 gCi [35S]methionine (1100
Ci/mmol, 5.4 mCi/ml; Amersham) were added to the cells for a 1 h pulse. At the end of the pulse period, the ceils
were washed with 1 ml PBS and dissolved in 200 gl 0.02 M-NaOH plus 1~ SDS and 200 gl sample buffer (62-5 mMTris-HC1 pH 6.8, 2 ~ SDS, 0.1 M-dithiothreitol, 17~ glycerol and 0.024~ bromophenol blue as indicator). Each
sample was sonicated to reduce viscosity and heated to 90°C for 5 min. Ten ktl was applied to a 15~
polyacrylamide gel and run overnight at 30 V. Fluorography of the gel was carried out with 2,5-diphenyloxazole at
20~o (w/w) in DMSO. The dried gels were exposed using XS-5 X-ray films (Kodak). Ten gl more of each sample
was precipitated with 1 ml 10~ TCA and filtered through GF/C glass fibre filters in a Millipore apparatus.
Toluene POPOP PPO (2-5 ml) was added to the dried filters and the radioactivity retained was estimated in an
Intertechnique scintillation spectrometer.
Measurement of S6Rb÷ content. HeLa cells grown in E4D10 medium were placed in 280 gl of a mixture of
methionine-free E4D1 medium :E4D 10 medium (3 : 1), 0.2 gCi 86Rb+ (1 mCi/ml; Amersham) was added, and the
cells were incubated for 17 h at 37 °C. Virus infection was then carried out while the 86Rb+ concentration was
maintained constant. At the times indicated, the cells were pulse-labelled with 0.14 gCi [35S]methionine. After 1 h
incubation, the medium was removed and the cells were washed three times with 1 ml PBS, and 0.5 ml 5 ~ TCA
was added to extract the 86Rb+ from the cells. The radioactivity of 0.4 ml of the TCA extract was determined by
estimating the Cerenkov radiation in a liquid scintillation counter. The level of protein synthesis was estimated in
parallel cultures as described above.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
-
2163
Translation regulation in virus-infected cells
HeLa cells
Time
I 3 5 7 9 1| 1 3 1 5 1 7 1 9 2 1 2 3
I
(h p.i.)
2
//
|(a)'
'
'
~0~-
'
'
o/~..,~,, o
/
.
/,
.... , \
°%, / ", ~,,
×
",.__]
",{
-o,--
4
L cells
I
Time
x
I 3 5 7 911131517192123
~0
(h p.i.)
I
I
t
I
I
o
I 04 tJ
.E
-12
3~
~
ez
:~
,, ..6
30
:'"
~
~o~O \
/o
\
,,
o
'e
4 "--o._
~
~'.
°~
2
0
0
I
4
I
8
I
I
° - - o - -o
I
12
16
20 24
Time (h)
Fig. 1. Kinetics of protein synthesis ( O ) and 86Rb+ content ( 0 ) in different reovirus-infected cells.
Cells were grown on 24-well Linbro dishes. Time of reovirus infection (20 p.f.u./cell) was considered as
- 1 h. Proteins were labelled by 1 h pulses with 5 IXCi [35S]methionine and processed as indicated in
Methods. The S6Rb+ content was estimated in parallel cultures also as described in Methods. Bands
corresponding to reovirus proteins (2, Ix, a) are indicated. (a) HeLa cells; (b) L cells.
RESULTS
Protein synthesis and 86Rb+ content in reovirus-infected cells
The reovirus genome comprises ten different pieces of dsRNA, each one coding for a single
protein (Joklik, 1981). These different proteins have been classified into groups according to
size. Infection of HeLa cells with reovirus type 3 led to massive synthesis of reovirus proteins
starting from about 6 h post-infection and continuing at very high levels for several hours. It is
striking that infection did not lead to inhibition of total protein synthesis as occurs with most
animal viruses, but instead total protein synthesis was stimulated almost 100~o compared to
uninfected cells. Also, cellular protein synthesis continued unabated for long periods of time,
indicating that cellular and viral protein synthesis co-exist (Fig. 1). However, infection of L cells
by reovirus interfered with cellular protein synthesis from about 10 h post-infection, and from 15
h onwards the cell translated viral m R N A s exclusively (Fig. 1). Analysis of membrane
permeability to monovalent cations in these cells indicated that a close parallel exists between
variations in protein synthesis and modifications in S6Rb+ content. For HeLa cells, there was an
increase in S6Rb+ until 11 h post-infection, followed by a gradual decline, but even at 20 h postinfection the S6Rb+ content was similar to that in uninfected cells (Fig. 1). Analysis of 86Rb+
uptake by very short pulses (15, 30 and 60 s) indicates a rapid increase in the uptake of this
cation, which was maximal (250~ of control) at 11 h post-infection (results not shown).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
2164
M. MUI~IOZ, M. A. ALONSO AND L. CARRASCO
(a)
f
Timep.i.
reovirus (h)
(b)
I
16 17 18 19 20 21 23 24
I
I
17 18 19 20 21 23 24
Fig. 2. Effect of VSV superinfection on protein synthesis in reovirus-infected HeLa cells. Cells grown
on 24-well Linbro dishes were infected with reovirus (20 p.f.u./cell) at - 1 h. Superinfection with VSV
(100 p.f.u./cell) was at + 15 h. Bands corresponding to VSV (L, G, N, NS, M) and reovirus (2, ~t, a)
proteins are indicated. (a) Reovirus-infected cells; (b) reovirus-infected cells superinfected with VSV at
+15h.
Theoretically, the higher uptake of this cation should be compensated by a higher release of
sodium ions, since at that time there is no loss of other cellular metabolites. The behaviour of
reovirus-infected L cells was similar to that of HeLa cells during the first 13 h of infection. After
that time, a drastic release of 86Rb+ occurred, accompanied by a cessation of synthesis of several
reovirus proteins. However, the synthesis of other reovirus proteins was particularly resistant to
these ionic modifications (Fig. 1).
Protein synthesis in HeLa cells infected with reovirus and superinfected with VSV, poliovirus or
E M C virus
According to the model proposed by Skup and co-workers (Skup & Millward, 1980; Skup et
al., 1981) for regulation of translation in reovirus-infected cells, a modification in the proteinsynthesizing apparatus late in reovirus infection takes place, thus preventing the cell from
translating capped m R N A s . To test this, reovirus-infected HeLa cells were superinfected late in
infection with VSV, a virus that possesses typical capped m R N A , after which the synthesis of
VSV proteins was analysed. Fig. 2 shows that VSV m R N A s were translated in these doubleinfected cells, suggesting that the protein-synthesizing machinery is in fact able to translate
capped m R N A s . As regards shut-offof protein synthesis by VSV in this system, it is worth noting
that cellular m R N A translation was shut down before reovirus protein synthesis. Twenty h after
reovirus infection, which corresponds to 5 h after VSV superinfection, no cellular proteins were
made, and there was then a significant decrease in reovirus protein synthesis (Fig. 2). Since
reovirus m R N A is a weaker competitor than cellular m R N A (Walden et al., 1981), this result
does not support the idea that the interference of VSV with translation is carried out by
competition with preexisting m R N A s .
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
Translation regulation in virus-infected cells
2165
Poliovirus added at + 1 h
I
Time p.i.
reovirus (h)
I
Guanidine
i
+ Guanidine
I
34
f
1
567810
34567810
41!
o
I
I
I
.~~...~ (a)_
×2
o--
6.1
I
Poliovirus added at + 15 h
!
1
-
Time p.i.
I
reovirus (h) 17
Guanidine
18 19 20212224
I
J
+ Guanidine
17181920212224
I
~0
i
5
o\o,x,k
8 "11 k._._
._=
"~ 6
I
•
I °-"-r~o
7
9
[
I
I
11
(b)
I
-
54
2
411
I
I
I
17
19
21
23
25
Time after reovirus infection (h)
Fig. 3. Effect of guanidine on protein synthesis in reovirus-infected HeLa cells superinfected with
poliovirus. Cells grown on 24-wellLinbro dishes were infected with reovirus (20 p.f.u./cell) at - 1 h, and
superinfected with poliovirus (75 p.f.u./cell) at either (a) + 1 or (b) + 15 h. Cells were treated with
guanidine (3 m~,l) where indicated from the beginning of poliovirus infection until the end of the
labelling period. Bands corresponding to some poliovirus (NCPVla, NCPVlb, NCPV2, VPO, VP3)
and reovirus (2, p., a) proteins are indicated. O, Untreated cells; Q, guanidine-treated cells.
The translation of uncapped m R N A s in reovirus-infected cells was analysed by superinfection with two different animal viruses, poliovirus and E M C viruses. Both are thought to block
protein synthesis by different mechanisms (Jen et al., 1980; Detjen et al., 1981). According to the
model suggested for reovirus-infected L cells, the translation of reovirus uncapped m R N A s is
blocked early during infection, and thus is only possible in the late phase (Skup & Millward,
1980; Skup et al., 1981). Fig. 3 and 4 indicate that this does not hold for picornavirus m R N A
translation in reovirus-infected HeLa cells, because poliovirus and E M C virus m R N A s were
translated both early and late during infection. Poliovirus protein synthesis is blocked in
interferon-treated cells (Mufioz & Carrasco, 1983, 1984) and also by the presence of guanidine
(Fig. 3) (Lacal & Carrasco, 1983). However, even though viral protein and R N A synthesis is
profoundly inhibited under those conditions, the shut-off of protein synthesis still occurs
(Mufioz & Carrasco, 1983; Mufioz et al., 1983). Analysis of the blockade by poliovirus of host
and reovirus protein synthesis late in reovirus infection of HeLa cells and in cells treated with
guanidine leads to a number of conclusions. First, both host and reovirus translation are blocked
by poliovirus infection in control cells, but host protein synthesis is more sensitive. This raises
the question that if reovirus and poliovirus block protein synthesis by a mechanism similar to
that already suggested (Skup & Millward, 1980; Skup et al., 1981) why is reovirus protein
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
2166
M. MUI~OZ, M. A. ALONSO AND L. CARRASCO
EMC virus superinfection
h after reovirus infection
Timep.i.
EMC virus (h)
1 3 4 5 6
+llh
+lh
Control
I
I
13456
I
I
I
1 3456
A
D
E
e, F
Y
I
I
I
I
I
I
I
2
I
3
I
4
5
I
~3
x
¢-~
o
¢x
.=.
2-0
1
I ~
.
6
Time after EMC virus infection (h)
Fig. 4. Protein synthesis in reovirus-infected HeLa cells superinfected with EMC virus. Cells grown on
24-well Linbro dishes were infected with reovirus (10 p.f.u./cell) at - 1 h, and superinfected with EMC
virus (100 p.f.u./cell) at either + 1 (O) or + 11 (Ak)h. Cells infected with EMC virus only were taken as
control (0). Bands corresponding to some EMC virus (A, D, E, e, ct, ),, H, I) and reovirus (2, ~t, a)
proteins are indicated.
synthesis inhibited? In addition, we know that E M C virus and poliovirus translation are
compatible in double-infected cells (Alonso & Carrasco, 1982a). On the other hand, inhibition
of poliovirus replication by means of guanidine still allows the shut-off of protein synthesis to
take place. However, it is particularly clear in guanidine-treated cells that the inhibition of host
protein synthesis occurs before the inhibition of reovirus protein synthesis. Moreover, not all
reovirus proteins behave in the same way, because some of them are more resistant to inhibition
than others. This result is difficult to explain if we assume that poliovirus destroys a factor
involved in cap recognition, necessary for the translation of capped reovirus m R N A s (Rose et
al., 1978). The pattern of translation after E M C virus superinfection was similar, i.e. E M C virus
protein synthesis occurred early and late during reovirus infection of H e L a cells, and shut-offof
host protein synthesis occurred before that of reovirus protein synthesis (Fig. 4). Three h after
E M C virus superinfection of reovirus-infected H e L a cells (14 h post-infection) no host
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
Translation regulation in virus-infected cells
2167
EMC virus superinfection at + 1 h
1
HEp-2
I
3T6
I
I
Time p.i.
4689
reovirus (h)
Vero
I
4689
I
BHK-21
I
4689
I
L
I
4689
I
l
46810
:
.... , .
~ - -
y
7:
-
I
EMC virus superinfection at + 11 h
Time p.i.
reovirus (h)
HEp-2
r - - I
14161819
3#
3T6
[ - - ]
14161819
:-:~
Vero
I
I
14161819
BHK-21
i
I
14161819
le~;'
'Z
.....
'
L
I
I
14161819
IilU~ z #
"I
Fig. 5. Effect of EMC virus superinfection on protein synthesis in different reovirus-infected
mammalian cells. Cells grown on 24-wellLinbro dishes were infected with reovirus (20 p.f.u./cell) at - 1
h, and superinfected with EMC virus (100 p.f.u./cell) at either + 1 or + 11 h. Bands corresponding to
EMC virus (A, D, E, e, F, a, y, H, I) and reovirus (2, ~t, a) proteins are indicated.
translation was evident, whereas reovirus protein synthesis continued unabated. This indicates
once again that E M C virus does not block protein synthesis by competition for a messagediscriminatory factor.
Comparison of different cell lines infected with reovirus and superinfected with E M C
virus or V S V
The inhibition of host translation after reovirus infection differed according to the cell type
analysed. For some cells like HeLa (Fig. 1) or SC1 (Walden et al., 1981) there was hardly any
inhibition of cellular protein synthesis, whereas for others like L cells or B H K cells (Fig. 1) the
blockade of host translation was noticeable. The model suggested by Skup and Millward was
based on experiments in L cells (Skup & Millward, 1980; Skup et al., 1981). It was therefore of
interest to compare the behaviour of different cell lines infected with reovirus, as regards the
translation of capped or uncapped m R N A s both early and late during infection. Since poliovirus
only infects cells of simian origin, we chose E M C virus as representative of a virus with an
uncapped m R N A and VSV to analyse capped m R N A translation in cells superinfected with
these viruses. Fig. 5 shows that, on superinfection with E M C virus of a variety of cell lines
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
2168
M. MUI~IOZ, M. A. ALONSO A N D L. C A R R A S C O
VSV superinfection at + 1 h
HEp-2
I
Time p.i.
4689
reovirus (h)
3T6
1
I
Vero
I
I
4689
BHK-21
I
L
I - - ]
4689
4689
46810
--L
--G
--N
--NS
--M
VSV superinfection at + 11 h
I
I
HEp-2
I
Time p.i.
14161819
reovirus (h)
3T6
I
I
14161819
Vero
I
I
14161819
BHK-21
I
I
14161819
L
I
I
14161819
I
L
N
G~--- NS
M
Fig. 6. Effect of VSV superinfection on protein synthesis in different reovirus-infected mammalian
cells. Cells grown on 24-wellLinbro dishes were infected with reovirus (20 p.f,u./cell) and superinfected
with VSV (100 p.f.u./cell) at either + 1 or + 11 h. Bands corresponding to VSV (L, G, N, NS, M) and
reovirus (,~, ~t, ~) proteins are indicated.
previously infected with reovirus, expression of E M C virus proteins occurred early during
infection. This leads to the conclusion that in none of the cell lines tested, including L cells, was
there any restriction of translation of uncapped E M C virus m R N A . On the other hand, in some
of the cell lines tested, such as L, HEp-2 and BHK-21 cells, no E M C virus proteins were detected
late in infection. This might be either because this uncapped m R N A is not translated in these
cells or, more likely, because viral replication cannot occur in these cells in the late phase of
reovirus infection.
Similarly, VSV protein synthesis was observed in all cell lines analysed early in infection at
levels comparabLe to control uninfected ceils, but once again no VSV proteins were detected late
during reovirus infection of L cells, whereas some inhibition was observed in HEp-2 and BHK21 cells (Fig. 6). These findings that capped m R N A s are translated late in infection in some cell
lines and that uncapped EMC virus m R N A is translated early in reovirus infection in all cell
lines tested do not support the idea of changes in the protein-synthesizing apparatus in every
type of reovirus-infected cell, although it might operate in L cells if it is proven that the
restriction of VSV replication is at the translational level.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
Translation regulation in virus-infected cells
2169
DISCUSSION
Two different models have been put forward to account for the regulation of translation in
reovirus-infected cells. One suggests that early during reovirus infection the infected cells
preferentially translate capped mRNAs, whereas in the late phase only uncapped m R N A s are
translated. The reason for this specificity could be that the protein-synthesizing machinery is
modified perhaps in a way similar to that suggested for poliovirus-infected cells (Skup et al.,
1981). Indeed, cell-free systems obtained late in infection from reovirus-infected cells translate
uncapped m R N A s exclusively, a result very similar to that obtained for poliovirus-infected cells
(Skup et al., 1981). However, the translation of capped viral m R N A s in intact poliovirus or
reovirus-infected cells occurs during the course of superinfection (Alonso & Carrasco, 1982b).
This suggests that the results in vitro cannot be directly equated with the situation in vivo. The
other model suggested for the regulation of translation after reovirus infection indicates that
viral and cellular m R N A s compete for a message-discriminatory factor which is present in the
cell in limiting amounts (Walden et al., 1981; Brendler et al., 1981a, b; Ray et al., 1983).
Although host m R N A s are better competitors than reovirus mRNAs, viral protein synthesis
occurs because the viral m R N A s are made in larger quantities (Walden et al., 1981). If this were
so, then superinfection with EMC virus or VSV should shut down reovirus protein synthesis
more effectively than host translation. Clearly, the results obtained in doubly infected cells show
that the contrary is true, i.e. host translation is more sensitive to inhibition than reovirus protein
synthesis. In fact, after EMC virus or VSV superinfection of reovirus-infected cells, there was a
drastic decline of total protein synthesis which is hardly explicable by weak m R N A s replacing
stronger mRNAs, as the competition model suggests.
As regards the possibility that reovirus-infected cells exclusively translate capped m R N A s
early during infection and switch to a cap-independent mechanism at late times, the
experiments reported in the present work suggest that this is not so for all cell lines analysed.
Thus, mouse cells such as L and 3T6 cells efficiently translate uncapped EMC virus RNA early
during reovirus infection. It might still be argued that the lack of ability of L cell extracts to
translate uncapped m R N A only holds for reovirus m R N A s and not for picornavirus RNA.
Besides, HEp-2, BHK-21 and L cells did not efficiently support the replication of EMC virus or
VSV when they were superinfected late in the reovirus cycle. The argument that in these cell
lines there is less VSV protein synthesis because these cells only translate uncapped reovirus
m R N A s needs to be substantiated by experiments designed to measure the step of VSV
replication inhibited late during reovirus infection of those cells.
An interesting finding was that the 86Rb+ content paralleled the changes observed in protein
synthesis both in HeLa and L cells infected with reoviruses. For L cells there was a marked
decrease in total protein synthesis from 13 h post-infection, at the same time as a decrease in
86Rb+ ions occurred. This indicates a loss of potassium ions from the cellular cytoplasm and
most probably entry of sodium ions. Curiously enough, the synthesis of some reovirus proteins
was more resistant than others to these ionic modifications.
The excellent technical assistance of Ms M. A. R a m o s is acknowledged. A.M. was the holder of a FIS
fellowship. This work was supported by grants from C A I C Y T and Fundaci6n Cientifica de la Asociaci6n
Espafiola contra el Cancer.
REFERENCES
ALONSO, M. A. & eARRASeO, L. (1982a). Protein synthesis in HeLa cells double-infected with encaphalomyocarditis
virus and poliovirus. Journal of General Virology 61, 15-24.
ALONSO, M. A. & eARRASeO, L. (1982b). Translation of capped viral m R N A s in poliovirus-infected HeLa cells.
EMBO Journal 1, 913-917.
BABLANIAN, R. (1972). Depression of macromolecular synthesis in cells infected with guanidine-dependent
poliovirus under restrictive conditions. Virology 47, 255-259.
BRENDLER, T., GODEFROY-COLBURN,T., CARLILL, R. D. & THACH, R. E. (1981 a). The role of m R N A competition in
regulating translation. II. Development of a quantitative in vitro assay. Journal of Biological Chemistry 256,
11747 11754.
BRENDLER, T., GODEFROY-COLBURN,T., YO, S. & THACH,R. E. (1981 b). The role of m R N A competition in regulating
translation. Ill. Comparison of in vitro and in vivo results. Journal of Biological Chemistry 256, 11755-11761.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56
2170
M. M U I ~ O Z , M. A. A L O N S O A N D L. C A R R A S C O
CARRASCO,L. (1977). The inhibition of cell functions after viral infection. A proposed general mechanism. FEBS
Letters 76, 11-15.
CARRASCO,L~ & SMITH, A. E. (1976). Sodium ions and the shut-offof host cell protein synthesis by picornaviruses.
Nature, London 264, 807-809.
CARRASCO,L. & SMITH, A. E. (1980). Molecular biology of animal virus infection. Pharmacology and Therapeutics 9,
311-355.
COLLINS, F. D. ~ ROBERTS,W. R. (1972). Mechanism of mengovirus induced cell injury in L cells: use of inhibitors of
protein synthesis to dissociate virus-specific events. Journal of Virology 10, 969 978.
DETJEN, B. M., JEN, G. & THACH, R. E. (1981). Encephalomyocarditis viral R N A can be translated under conditions
of poliovirus-induced translation shut-off in vivo. Journal of Virology 38, 777 781.
DETJEN, B. M., WALDEN, W. E. & THACH, R. E. (1982). Translation specificity in reovirus-infected mouse fibroblasts.
Journal of Biological Chemistry 257, 9855 9860.
GODEFROY-COLBURN, T. & THACH, R. E. (1981). The role of m R N A competition in regulating translation. IV.
Kinetic model. Journal of Biological Chemistry 256, 11762-11773.
GOLINI, F., THACH,S. A., BIRGE, C. H., SAFER, B., MERRICK,W. C. & THACH,R. E. (1976). Competition between cellular
and viral m R N A s is regulated by a messenger discriminatory initiation factor. Proceedings of the National
Academy of Sciences, U.S.A. 73, 3040-3044.
HOLLAND, J. J. (1964). Inhibition of host cell macromolecular synthesis by high multiplicities of poliovirus under
conditions preventing virus synthesis. Journal of Molecular Biology 8, 574~581.
JEN, G., DETJEN, a. M. & TrfACH, R. E. (1980). Shut-offof HeLa cell protein synthesis by encephalomyocarditis virus
and poliovirus: a comparative study. Journal of Virology 35, 150-156.
~OKUK, w. K. (1981). Structure and function of the reovirus genome. Microbiological Reviews 45, 483 501.
LACAL,J. C. & CARRASCO,L. (1982). Relationship between m e m b r a n e integrity and the inhibition of host translation
in virus-infected m a m m a l i a n cells. Comparative studies between encephalomyocarditis virus and poliovirus.
European Journal of Biochemistry 127, 359-366.
LACAL, J. C. & CARRASCO,L. (1983). Modification of m e m b r a n e permeability in poliovirus-infected HeLa cells:
effect of guanidine. Journal of General Virology" 64, 787-793.
LUCAS-LENARD, J. M. (1979). Inhibition of cellular protein synthesis after virus infection. In The Molecular
Biology of Picornaviruses, pp. 73-101. Edited by R. P6rez-Bercoff. New York & London: Plenum Press.
MUr~OZ, A. & CARRASCO,L. (1981). Protein synthesis and m e m b r a n e integrity in interferon-treated HeLa cells
infected with encephalomyocarditis virus. Journal of General Virology 56, 153-162.
MUI~OZ, A. & CARRASCO,I-. (1983). Effect of interferon treatment on blockade of protein synthesis induced by
poliovirus infection. European Journal of Biochemistry 137, 623-629.
MUfJOZ, A. & CARRASCO,L. (1984). Action of h u m a n lymphoblastoid interferon on HeLa cells infected with R N A containing animal viruses. Journal of General Virology 65, 377 390.
MUF;OZ, A., HARVEY,R. & CARRASCO,L. (1983). Cellular R N A is not degraded in interferon-treated HeLa cells after
poliovirus infection. FEBS Letters 160, 87 92.
RAY, B. K., BRENDLER, T. G., ADYA, S., DANIELS-McQUEEN,S., MILLER, J. K., HERSHEY, J. W. B., GRIFO, J. A., MERRICK,
W. C. & THACH, R. E. (1983). Role of m R N A competition in regulating translation: further characterization of
m R N A discriminatory initiation factors. Proceedingsof the National Academy of Sciences, U.S.A. 80, 663-667.
ROSE, J. K., THRACHSEL,H., LEONG, K. & BALTIMORE,D. (1978). Inhibition of translation by poliovirus: inactivation
of a specific initiation factor. Proceedings of" the National Academy of Sciences, U.S.A. 75, 2732-2736.
SKUP, D. & MILLWARD, S. (1980). Reovirus-induced modification of cap-dependent translation in infected cells.
Proceedings of the National Academy of Sciences, U.S.A. 77, 152-156.
SKUP, D., ZARBL, H. & MILLWARD,S. (1981). Regulation of translation in L cells infected with reovirus. Journal of
Molecular Biology 151, 35 55.
STEINER-PRYOR,A. & COOPER, P. D. (I 973). Temperature-sensitive poliovirus m u t a n t s defective in repression of host
protein synthesis are also defective in structural protein. Journal of General Virology 21, 215-225.
WALDEN, W. E., GODEFROY-COLBURN,T. & THACH, R. E. (1981). The role of competition in regulating translation. I.
Demonstration of competition in vivo. Journal of Biological Chemistry 256, 11739-1 1746.
(Received 28 January 1985)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 03:14:56