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Journal of Virology, August 1999, p. 7070-7076, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Resistance of Ribosomal Protein mRNA Translation to
Protein Synthesis Shutoff Induced by Poliovirus
Beatrice
Cardinali,1
Lucia
Fiore,2
Nadia
Campioni,1
Alessandra
De
Dominicis,1 and
Paola
Pierandrei-Amaldi1,*
Istituto di Biologia Cellulare
CNR,1 and Laboratorio di Virologia,
Istituto Superiore di Sanita',2 Rome, Italy
Received 3 February 1999/Accepted 27 April 1999
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ABSTRACT |
Poliovirus infection induces an overall inhibition of host protein
synthesis, although some mRNAs continue to be translated, suggesting
different translation requirements for cellular mRNAs. It is known that
ribosomal protein mRNAs are translationally regulated and that the
phosphorylation of ribosomal protein S6 is involved in the regulation.
Here, we report that the translation of ribosomal protein mRNAs resists
poliovirus infection and correlates with an increase in
p70s6k activity and phosphorylation of ribosomal
protein S6.
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TEXT |
Poliovirus infection results
in a drastic shutoff of cellular protein synthesis, accompanied by a
selective production of viral proteins (11, 54). This is
achieved mostly at the level of translation by specific impairment of
the cap-dependent initiation step (17, 28). In fact, the
mRNA of poliovirus, and that of other picornaviruses, is uncapped
and characterized by a long and structured 5' untranslated region
(5'UTR) where an internal ribosome entry site (IRES) can promote
cap-independent translation initiation (19, 22, 43). One of
the mechanisms responsible for the inhibition of cap-stimulated
translation involves the modification and inactivation of the
translation initiation factor eIF4F, due to the cleavage of the eIF4G
subunit (11). On the other hand, the eIF4G cleavage products
can facilitate the translation initiation of viral RNAs, mediated by an
IRES, and of uncapped cellular RNAs (41). Although most of
the host protein synthesis is inhibited in poliovirus-infected cells,
the translation of some cellular mRNAs occurs. They include the
heat shock protein (HSP) mRNAs and the immunoglobulin heavy-chain
binding protein, c-myc, and eIF4G mRNAs, which use the mechanisms
of internal initiation (25, 39, 50). The cellular
modification induced by viral infection to cellular protein synthesis
can help to identify the mechanisms that normally control mRNA translation.
We were interested in the regulatory mechanisms that control the
translation of ribosomal protein (rp) mRNAs
(rp-mRNAs) (1, 37). It is known that the
translation of rp-mRNAs is regulated by elements contained in the
5'UTR of rp-mRNAs and, in particular, by a typical terminal
oligopyrimidine segment (29, 34). Putative transacting
factors can bind the 5'UTR of rp-mRNAs in mammalian and
Xenopus cells (5, 26), where they were identified
as the La protein and the cellular nucleic acid binding protein
(44, 45). Furthermore, it was reported that in
mitogen-stimulated cells, the efficiency of translation of mRNAs
carrying a 5'-terminal pyrimidine tract is mediated by the activity of
p70S6k, the kinase responsible for the phosphorylation of r
protein S6 (4, 23, 24, 55).
In this study, we have investigated the behavior of the class of
rp-mRNAs under the translational conditions caused by poliovirus infection, in order to obtain information on the mechanisms that control their translation.
Translation of rp-mRNAs during poliovirus
infection.
We analyzed the mRNA distribution between polysomes
and messenger ribonucleoprotein particles (mRNPs) in mock-infected
and poliovirus-infected HEp-2 cells. Cells were infected with the poliovirus type 1 Mahoney strain and incubated for 4 h. Extracts corresponding to one plate of cell culture from mock-infected and
poliovirus-infected cells were fractionated by sucrose gradient centrifugation, and the RNA was extracted from the fractions. Amounts
corresponding to the same volumes of gradient fractions were analyzed
by Northern blotting as previously described (32). A
representative example of these experiments is given in Fig. 1, where the polysome-mRNP
distribution of rp-mRNAs is compared to that of 
-actin mRNA,
a control mRNA subjected to shutoff. It should be noticed that
in these experiments, only the distribution of the mRNAs
along the gradients should be compared between mock-infected and
infected cells and not the absolute amount of RNA. In mock-infected cells, about 70 to 80% of the mRNAs analyzed were loaded
onto polysomes to be actively translated. In infected cells, -actin mRNA was mostly displaced to mRNPs at the top of the
gradient, as expected, while a large part of the L4, L32, and L11
rp-mRNAs was still associated with polysomes. However, these
rp-mRNAs were associated with small polysomes, indicating that in
infected cells, translation initiation might be less efficient than in
uninfected cells. To obtain further information about the translational
behavior of rp-mRNAs at different infection times, we
analyzed the polysome-mRNP distribution of L4, L32, L11, and
-actin mRNAs at 90 min and 4 h after infection. Figure
2A shows graphically that, compared with mock-infected cells, the distributions of the rp-mRNAs and -actin were soon quite different. At 90 min after infection, polysome-associated -actin mRNA started decreasing and the
dislocation of this mRNA to the top of the gradient reached 90%
within 4 h. On the contrary, 90 min after infection, about 60% of
the L4, L32, and L11 mRNAs was still associated with large
polysomes and after 4 h they remained associated with polysomes
which, however, were smaller. Since gradient analysis is intended to
show the translational activity of the mRNAs and not to quantify
their absolute amount, quantitative aliquots of each extract at
different times of infection were taken before gradient loading for
total RNA analysis by Northern blot hybridization to different probes. The hybridization signals were quantified by comparison with 5S rRNA,
which is structured in the ribosomes and therefore is expected to be
fairly stable. Figure 2B shows that the level of all the mRNAs
analyzed does not change appreciably up to 90 min and, with minor
differences, decreases by about 30 to 35% at 4 h, indicating that
rp-mRNAs and -actin mRNA are subjected to similar rates of
degradation.
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FIG. 1.
Polysome-mRNP distribution of mRNA in
mock-infected and poliovirus-infected cells. HEp-2 cells were grown at
37°C in Eagle's minimum essential medium supplemented with 10%
fetal calf serum. When cells reached 80% confluence, the Mahoney
strain of poliovirus type 1 was added at a multiplicity of infection of
50 PFU per cell. A 9-cm-diameter plate culture of mock-infected and
infected cells was lysed at 4 h postinfection (10 mM NaCl, 10 mM
MgCl2, 1% Triton X-100, 1% Na-deoxycholate, 1 mM
dithiothreitol, 10 mM Tris-HCl, pH 7.4) to prepare cytoplasmic extracts
(32). Cycloheximide, often used to prevent polysome runoff,
was not added prior to extract preparation, as this drug sometime
creates problems (38). Polysome protection can also be
obtained by quickly preparing the extract and loading the sample on the
gradient under strict temperature control. The extracts were separated
on 15 to 50% sucrose gradients in gradient buffer (0.1 M NaCl, 10 mM
MgCl2, 30 mM Tris-HCl, pH 7). Gradient fractions were
collected while the optical density profile at 260 nm was monitored
(top), and the RNA was prepared by protease K-SDS-phenol extraction
(32) of the fractions. The RNAs from equal volumes of
mock-infected and infected gradient fractions were loaded onto two gels
and analyzed by Northern blotting and autoradiography as previously
described (32). Each filter was subsequently hybridized to
probes for rp-L4 (2), rp-L32 (10), rp-L11
(12), and -actin (6) mRNAs to obtain a
reliable comparison of the distribution of the various RNAs along the
same gradient. Probes were prepared by the random primer technique.
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FIG. 2.
Time course of polysome-mRNP distribution and
accumulation of mRNAs during poliovirus infection. (A)
Mock-infected and poliovirus-infected cells were processed at the times
indicated as described in the legend to Fig. 1. Northern blots were
subsequently hybridized with rp-L4, rp-L11, rp-L32, and -actin
probes. Measurement of radioactivity, reported as the percentage of
mRNA in each fraction, was done by PhosphorImager (Molecular
Dynamics) analysis. The optical density profiles of the sucrose
gradients were monitored at 260 nm (top), and the positions of the 80S
monomers are indicated. These experiments were performed at least three
times, and the results were consistently similar. (B) One tenth of each
extract, prepared at the indicated time postinfection, was taken before
gradient loading. The RNA was extracted, and equivalent amounts were
analyzed by Northern blotting as described in the legend to Fig. 1. The
filters were subsequently hybridized to rp-L4, rp-L32, rp-L11,
-actin, and 5S RNA probes (46). Measurement of
radioactivity was done by PhosphorImager (Molecular Dynamics) analysis,
and the values obtained were normalized to the signal of the 5S rRNA
probe. The level of each mRNA is expressed as a percentage of the
amount measured in mock-infected cells (c).
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To check the activity of polysome-associated mRNA after poliovirus
infection, at 90 min and 3.5 h postinfection,
[
35S]methionine-[
35S]cysteine was added for
30 min to HEp-2 cells to label synthesized
r proteins. Thirty minutes
of radioactive precursor administration
was reasonable for detection of
accumulated labeled proteins and
to overcome the expected effect of r
protein instability due to
decreased rRNA synthesis, as will be
discussed later. Considering
the short labeling time and the fact that
more extract could not
be loaded onto the gel without pattern
distortion, only faint
spots can be expected on the two-dimensional
(2D) gel, in accordance
with data previously obtained with other
systems (
47). After
incubation, protein extracts were
prepared from mock-infected
and infected cells and analyzed on a 2D gel
optimized to resolve
ribosomal proteins (
58). Gels were
Coomassie stained and fluorographed.
Radioactive r proteins were
identified on the 2D gel by their
comigration with the purified human r
proteins included in the
sample (
58). A polysome gradient
experiment was performed with
an aliquot of the extracts from
mock-infected and infected labeled
cells to check that the RNA
distribution was as expected, namely,
as in Fig.
1 (data not shown). At
the same time, a protein labeling
experiment was performed to check the
pattern of total protein
synthesis during infection. Figure
3A shows that at 2 h postinfection,
the synthesis of r proteins is still efficient (arrowheads), in
agreement with the rp-mRNA engagement with polysomes described
above. However, at this time, the general inhibition of host cell
protein synthesis is already detectable, as shown by the pattern
of
total protein synthesis at different times after infection
(Fig.
3B).
Note also the remarkable shutdown of two non-r proteins
that can be
seen in Fig.
3A (arrows). At 4 h postinfection, r
protein labeling
is no longer detectable (data not shown). This
might be due to the
severe inhibition of rRNA synthesis during
poliovirus infection
(
7,
49) that becomes relevant with time,
thus preventing
accumulation of newly synthesized r proteins.
Indeed, it has been
observed in other systems that newly synthesized
r proteins become
unstable when rRNA needed for assembly is not
available (
8,
47,
58). It was recently reported that, following
herpes simplex
virus type 1 infection, r proteins continue to
be synthesized during
protein synthesis shutoff. However, in this
system, where 60% rRNA
synthesis persists in infected cells, r
proteins are stable,
as they can assemble with rRNA into ribosomes
(
52). To
ascertain rp-mRNA association with polysomes later
in infection as
well, some control experiments were carried out.
Cytoplasmic extracts
were prepared 4 h postinfection and treated
with EDTA to
dissociate polysomes. Compared to untreated infected
cells, a typical
rp-mRNA such as L4 was shifted by EDTA treatment
to the top of the
gradient, implying association with polysomes
(Fig.
3C, top and
middle). Similar results were obtained for the
other rp-mRNAs (data
not shown). Moreover, to show that polysome
association was due to
active translation, cells were treated
with pactamycin for 30 min at
3.5 h postinfection. This drug,
a translation inhibitor at the
initiation step, caused a decrease
in polysome size which resulted in
the accumulation of L4 mRNA
in the dimer and 80S fractions (Fig.
3C, bottom). The same occurred
to other rp-mRNAs (data not shown).
Rehybridization of the filters
with the viral probe showed a signal of
the expected size peaking
on fraction 3. This might represent the viral
particles, which
migrate close to, but never coincide with, the
rp-mRNAs (data
not shown). These results support the hypothesis
that rp-mRNAs
associate with polysomes, a view further
strengthened by the fact
that at 2 h postinfection, a similar RNA
distribution along the
gradient corresponds to protein synthesis
activity.
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FIG. 3.
Analysis of the activity of polysome-associated RNA in
infected cells by metabolic labeling of proteins and by EDTA and
pactamycin treatment. (A) HEp-2 cells were grown and infected as
described in the legend to Fig. 1. At 90 min postinfection,
mock-infected and infected cells were incubated for 30 min with
[35S]methionine-[35S]cysteine (Pro-mix;
Amersham; >1,000 Ci/mmol) at a concentration of 0.1 mCi/ml. Cells were
harvested in phosphate-buffered saline and homogenized in 0.5 ml of
ice-cold rp buffer (0.1 M NaCl, 1 mM MgCl2, 10 mM HEPES, pH
7.5), acid extracted (58), and processed by the 2D gel
electrophoresis method optimized to resolve r proteins, with the
exception that the second-dimension (2D) gel was 13% polyacrylamide
(58). A 200-µg sample of r proteins purified from HEp-2
ribosomes (58) was added as markers to an equal amount of
control (c) or infected-cell (i) protein extract (200 µg). Gels were
fluorographed and exposed to X-ray film for the same time. Arrowheads
point to some r proteins, and arrows point to non-r proteins. 1D, first
dimension. (B) HEp-2 cells, grown and infected as indicated above, were
labeled with Pro-mix (40 µCi/ml) for 10 min at the indicated times
after infection. Cells were lysed as described above, and 5 µg of
each extract was loaded onto an SDS-12.5% PAGE gel and
autoradiographed. Arrows point to viral proteins. Lane C, control. (C)
Untreated infected cells (top) were lysed at 4 h postinfection and
analyzed on a sucrose gradient as described in the legend to Fig. 1.
For the EDTA treatment (middle), cytoplasmic extracts, brought to a
concentration of 50 mM EDTA, pH 7.4, were incubated in ice for 5 min,
loaded onto sucrose gradients containing 10 mM EDTA instead of
magnesium, and analyzed as described in the legend to Fig. 1. To test
the effect of pactamycin (bottom), cells were incubated at 3.5 h
after infection with 30 ng of pactamycin per ml for 30 min, thus
reaching the 4-h infection time of untreated cells, and then processed
as described in the legend to Fig. 1. When the gradient fractions were
collected, the optical density at 260 nm was monitored. The profiles
are shown as a continuous line, and the 80S monomers are indicated by
the arrows. Northern blots were hybridized with an rp-L4 probe.
Measurement of radioactivity, reported as a percentage of the mRNA
in each fraction, was done by PhosphorImager (Molecular Dynamics)
analysis.
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From the results described above, it appears that rp-mRNAs are
fairly resistant to protein synthesis shutoff compared to
-actin
mRNA. Therefore, in spite of the complete cleavage of eIF4G which
already occurs 1 h after infection (Western blot in Fig.
4), rp-mRNAs
can still initiate
protein synthesis. Similarly, HSP mRNAs are
resistant to
poliovirus-induced shutoff and it has been proposed
that they may
utilize cap-independent initiation (
48). The limited
secondary structure of the HSP 5'UTR, as well as the short and
unstructured 5'UTR of rp-mRNAs, may determine a lower dependence
on
initiation factors compared to more-structured mRNAs (
9,
20,
53). In line with this, it has been reported that the
efficiency
of translation of rp-mRNAs is regulated independently
of the level
or activity of eIF4E (
51), whereas the selective
translational repression of mRNAs bearing extensive secondary
structure in the 5'UTR is relieved by the overexpression of this
factor
(
27). It was recently reported that eIF4GII, a functional
homolog of eIF4G (hence called eIF4GI), can persist longer in
poliovirus-infected cells, as shown by the fact that about 30%
of the
entire form still persists at up to 2 h after infection
(
14,
15). It can be argued that the rp-mRNA association with
polysomes and the r protein synthesis described here could be
sustained
by eIF4GII in infected cells. However, later in infection,
when
eIF4GII is completely cleaved, rp-mRNAs are still associated
with polysomes, suggesting that other elements are also involved.
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FIG. 4.
eIF4G cleavage in poliovirus-infected cells. Cytoplasmic
extracts from mock-infected (lanes c) and poliovirus-infected (lanes i)
cells were prepared at the times indicated as described in the legend
to Fig. 1. Aliquots of the cytoplasmic extracts were precipitated with
acetone for protein analysis by SDS-PAGE and Western blotting using the
anti-eIF4G antibody as previously described (18).
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Analysis of p70S6k activity and S6 phosphorylation
during poliovirus infection.
As mentioned above, a
relationship among translation of rp-mRNAs, activity of
p70S6k, and phosphorylation of r-protein S6 has been
reported (23, 24, 55). To investigate the state of
p70S6k activity after poliovirus infection, we performed in
vitro kinase assays by using p70S6k immunoprecipitated from
equal amounts of mock-infected and infected-cell extracts and 40S
ribosomal subunits as a substrate (16). As exemplified by
the experiment of Fig. 5A, the ability of
p70S6k to phosphorylate S6 was maintained and even
increased 1 h after infection, reaching a level of two- to
three-fold at 5 h. Similar results were consistently obtained by
either immunoprecipitation or direct incubation with 40S ribosomes of
equal amounts of control and infected-cell extracts. Although we cannot
determine whether this was due to higher p70S6k activity or
to an increased amount of it, the experiments reproducibly showed an
increase in the capacity of p70S6k to phosphorylate S6 in
infected cells. Then, to analyze S6 phosphorylation in vivo, cells were
labeled with [32P]orthophosphate for 90 min at 2.5 h
postinfection, thus reaching a total infection time of 4 h, when
the in vitro phosphorylating activity was still increasing (Fig. 5A).
Proteins were analyzed by 2D gel electrophoresis as previously
described (33), with the exception that in the second
dimension, a sodium dodecyl sulfate (SDS)-15% polyacrylamide gel
electrophoresis (PAGE) gel was used. The 2D gel electrophoresis
conditions were set up to map S6 phosphorylated forms, as indicated in
the legend to Fig. 5B. In this system, the hyperphosphorylated forms
migrate slower in the first dimension. Figure 5B shows two identical
gels loaded with extracts from
[32P]orthophosphate-labeled control and infected cells.
Compared to control cells, infected cells show a slight shift of the S6 spot toward the anode, as measured by the relative positions of the
radioactive S6 and stained, purified r proteins included in each
sample. A small decrease in hypophosphorylated forms and a small
increase in hyperphosphorylated forms can be seen. This finding,
observed in repeated experiments, suggests that in infected cells, the
level of S6 phosphorylation is maintained and indeed slightly
increased, compared to that in control cells, in line with the result
of the p70S6k kinase assay. Similarly, the activity of the
S6 kinase and S6 phosphorylation are stimulated following herpes
simplex virus type 1 infection (21, 35).
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FIG. 5.
p70S6k activity and S6 phosphorylation
during poliovirus infection. (A) S100 cytoplasmic extracts from
mock-infected (lane c) and poliovirus-infected (i) cells were prepared
at the times indicated in the presence of a phosphatase inhibitor as
previously described (40). Protein concentration was
determined by the Bio-Rad Protein Assay kit. Eight-microgram extract
samples were used for immunoprecipitation of p70S6k with M6
antibody, and the immunocomplex was assayed for in vitro kinase
activity in the presence of 40S ribosomes at an
A260 of 0.45 U as previously described
(16). Proteins were separated by SDS-PAGE and
autoradiographed. Phosphorylated S6 protein is indicated by the arrow.
(B) Mock-infected and poliovirus-infected cells (c and i, respectively)
were labeled at 2.5 h after infection in a phosphate-free medium
with 20 µCi of [32P]orthophosphate per ml for 90 min.
Ribosomes from labeled cells were purified, and ribosomal proteins were
extracted in the presence of 40 mM -glycerophosphate
(58). Proteins were analyzed by 2D electrophoresis as
previously described (33), with the exception that the
second dimension (2D) was an SDS-15% PAGE gel. A 200-µg sample of
unlabeled purified HEp-2 ribosomal proteins was added to each sample
before gel loading for Coomassie staining and used as position markers
in the gel. The positions of stained ribosomal proteins in the S6 area
are indicated by open circles. To standardize 2D gel electrophoresis
conditions and to map the positions of S6 phosphorylated forms,
preliminary experiments were carried out by loading gels with purified
40S subunits phosphorylated in in vitro kinase assays by extracts from
quiescent and serum-stimulated Swiss 3T3 cells (16). The
level of S6 phosphorylation by quiescent cell extracts was very low and
increased 20-fold after serum stimulation. Accordingly, an evident
shift in migration of S6 in the 2D gel was observed (data not shown).
1D, first dimension.
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It is known that both p70
S6k activation and the
phosphorylation of the initiation factor 4E binding protein (4E-BP1)
are mediated
by the mTOR-FRAP signalling pathway (
3,
31,
57). The different
phosphorylation state of 4E-BP1 affects eIF4E
activity, since
the dephosphorylated form can sequester eIF4E, thus
blocking cap-dependent
initiation (
30,
42). It has been
recently proposed that the
p70
S6k-4E-BP1 phosphorylation
pathway bifurcates immediately upstream
from p70
S6k
(
56). Interestingly, it has been found that 4E-BP1 becomes
dephosphorylated after poliovirus infection (
13) while our
data
indicate that p70
S6k activity and S6 phosphorylation
do not decrease but are maintained
and somewhat stimulated in infected
cells. If this is the case,
these observations might suggest that
poliovirus infection either
influences the p70
S6k-4E-BP1
kinase pathway downstream from the bifurcation or differentially
regulates specific
phosphatases.
In conclusion, we have identified a new class of cellular
mRNAs that, besides HSP (
39), and some
IRES-containing cellular
mRNAs recently analyzed with an
approach similar to the one used
in this study (
25), can be
translated in poliovirus-infected
cells. Moreover, we have shown that
the selective translation
resistance of rp-mRNAs correlates with
the maintenance of p70
S6k activity and with a small, but
consistent, phosphorylation increase
in r protein S6. It is hard to
believe that r protein synthesis
resistance is an advantage for the
virus, as rRNA synthesis is
inhibited and ribosome assembly is not
possible. It is more likely
that r protein mRNA translation can
continue because the conditions
required are still sufficient, in spite
of the drastic damage
to cellular translation initiation generated by
the infection.
This translation survival, that so far can be identified
as lower
dependence on initiation factors, may reveal a feature of the
normal mechanism governing rp-mRNA translation in the cell. It
is possible to speculate that this mechanism can be due to the
short
and typical rp-mRNA 5'UTR; to the utilization of specific
ribosomes
containing hyperphosphorylated S6, a feature that appears
to persist
under infection conditions; and to auxiliary factors
that specifically
bind the 5'UTR of rp-mRNAs (
44,
45). Interestingly,
one
of these binding factors, La protein, is known to have a positive
role in poliovirus RNA translation in vitro (
36).
Study of the
mechanisms that govern cellular mRNA resistance to
poliovirus-induced
shutoff of protein synthesis adds to our
knowledge of the cellular
response to viral infection and should
provide important clues
to understand the translational regulation of
mRNAs.
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ACKNOWLEDGMENTS |
We thank L. Carrasco for the anti-eIF4G antibody, G. Thomas for the
M6 anti-p70S6k antibody, and Pharmacia & Upjohn and M. Kleijn for pactamycin. We also thank F. Amaldi, G. Giannini, and F. Loreni for their critical reading of the manuscript.
This work was partially supported by the EC-DGXII Biotech Program and
by Progetto Strategico CNR "Posttranscriptional Controls of Gene Expression."
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FOOTNOTES |
*
Corresponding author. Mailing address: Istituto di
Biologia Cellulare CNR, Viale Marx 43, 00137 Rome, Italy. Phone:
39-06-86090353. Fax: 39-06-8273287. E-mail:
pierandrei{at}ibc.rm.cnr.it.
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Journal of Virology, August 1999, p. 7070-7076, Vol. 73, No. 8
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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