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Journal of Virology, September 2000, p. 8781-8784, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Protein Synthesis Shut-Off Induced by Influenza
Virus Infection Is Independent of PKR Activity
Thomas
Zürcher,
Rosa María
Marión, and
Juan
Ortín*
Centro Nacional de Biotecnología
(CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
Received 25 February 2000/Accepted 26 June 2000
 |
ABSTRACT |
The role of PKR activity in influenza virus-induced cell shut-off
was studied by infection of PKR+ or PKR
cell
cultures and metabolic labeling in vivo. No differences in the
synthesis of viral proteins or the decay of cellular
protein synthesis were observed. To investigate the relevance of the
inhibition of cellular pre-mRNA polyadenylation and
nucleocytoplasmic transport in virus-induced shut-off, we carried out
similar experiments with mutant viruses lacking C-terminal sequences of
NS1 protein. No differences in the shut-off induced by mutant versus
wild-type viruses were observed, indicating that these nuclear events
are not relevant for shut-off. The analysis of cytoplasmic mRNA
stability indicated that the accumulation of viral mRNA during the
infection correlated with the progressive decay of cellular mRNA, in
both the wild type and an NS1 deletion mutant.
| |
TEXT |
The influenza virus infection of
permissive cells leads to a progressive decline in the synthesis
of cellular proteins, a phenomenon known as cell shut-off.
Several steps in the gene expression program of the cell are altered by
influenza virus infection. Thus, the transport of cellular mRNA from
the nucleus to the cytoplasm is inhibited (18). This result
was first interpreted as a consequence of the cap-snatching activity of
the viral polymerase in the nucleus (18), and it was later
explained as the outcome of the inhibition of hnRNA polyadenylation
mediated by NS1 protein (24, 27). In influenza virus
infection, the translation machinery is utilized essentially to produce
viral proteins. Several observations may be relevant to explain this
fact. The cellular mRNAs present in the cytoplasm before the infection
are degraded during the infection cycle (2, 16) (see below).
Translation of cellular mRNAs is inhibited at both the initiation
and elongation steps (17), and viral mRNAs are
preferentially translated (12, 13), presumably as a
consequence of the activity of NS1 protein (6, 7, 22, 26).
The protein kinase PKR is one of the effectors of the interferon (IFN)
response. Its expression is activated by IFN, and its activity is
induced by double-stranded RNA (dsRNA). Activation of PKR involves its
autophosphorylation and leads to the phosphorylation of the 
subunit
of eukaryotic initiation factor 2 (eIF-2) and the subsequent
inhibition of protein synthesis (for reviews, see references
5 and 10). It has been argued
that influenza virus mRNAs are intrinsically "better
translators" than cellular ones, and the observed effect of NS1 in
this regard might be simply the consequence of blocking the activity of
PKR (21), since NS1 protein is also a dsRNA-binding protein.
To directly test this proposal and to evaluate the relevance of the
above-mentioned alterations of cell metabolism in the induction of cell
shut-off, we have studied the expression of cellular and viral genes in cells with a PKR gene knockout mutation and hence devoid of PKR activity.
Cultures of NIH 3T3 cells (PKR+) or the corresponding cells
mutated at the pkr gene (PKR) (28)
were infected with influenza virus (WSN strain) at a multiplicity of
infection of 5 to 10 PFU per cell and labeled by incorporation of
[35S]Met-Cys for 1 h at various times after
infection. Total cell extracts were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and the proteins were
visualized by autoradiography. The results are presented in
Fig. 1. No obvious differences in the
amounts of the viral proteins synthesized during the infection in
PKR+ and PKR cells were observed (see bands
labeled NP, M1, NS1, and NS2 in Fig. 1A). Likewise, the synthesis of
cellular proteins was inhibited progressively during the
infection to an extent and with a kinetics similar to those in both
PKR+ and PKR cells (see bands indicated
by stars in Fig. 1A and their quantification in Fig. 1B). To
verify that the PKR cells are devoid of PKR
protein, we carried out Western blot analyses with a
PKR-specific antiserum and extracts obtained from either
PKR+ or PKR cells. The results are
presented in Fig. 1C and confirm the correct phenotype of either
culture. These results indicate that whatever mechanism is responsible
for the induction of cell shut-off in influenza virus-infected cells,
it is independent of the activity of PKR.
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FIG. 1.
Effect of PKR expression on influenza virus-induced cell
shut-off. (A) Cultures of PKR+ or PKR NIH 3T3
cells were infected with the WSN strain of influenza virus at a
multiplicity of infection of 5 to 10 PFU per cell. At the times
indicated at the top of each lane, the cultures were labeled with
[35S]Met-Cys (200 µCi/ml) for 1 h and processed by
polyacrylamide gel electrophoresis and autoradiography. A selection of
the virus-encoded proteins is indicated to the right of each panel. The
stars point to prominent cellular proteins whose synthesis declined
during the infection. (B) The bands indicated by stars in panel A were
quantified by microdensitometry. The results have been normalized with
respect to the initial pulse period. (C) Total cell extracts were
prepared from cultures of PKR+ or PKR NIH 3T3
cells and analyzed by Western blotting with anti-PKR serum.
|
|
As indicated above, the expression of NS1 protein leads to the
inhibition of cellular pre-mRNA cleavage and polyadenylation by
interaction with the 30-kDa subunit of CPSF (24) and to the accumulation of pre-mRNA in the nucleus (27). In
addition, NS1 protein inhibits poly(A) elongation of mRNAs in the
nucleus and their export as a consequence of its interaction with
poly(A)-binding protein II (4). To investigate whether
such alterations play a role in the influenza virus-induced shut-off,
we took advantage of recently prepared mutant viruses that express
versions of NS1 protein containing deletions. These viruses express
short NS1 proteins containing either the 81 or 156 N-terminal amino
acids, and therefore they miss the C-terminal effector domain entirely (NS1-81) or partially (NS1-156). These two mutants grow in single-cycle infections as efficiently as wild-type virus, although the expression of late viral proteins is reduced due to a low accumulation of late
mRNAs. Their generation and phenotype will be described in detail
elsewhere. Since the interaction of NS1 protein with the 30-kDa subunit
of CPSF takes place through the C-terminal sequences of the former
(24), if such interaction is relevant for the inhibition of
cellular protein synthesis, then the C-terminally deleted NS1 proteins
would be predicted to show an affected shut-off phenotype. Cultures of
PKR+ or PKR NIH 3T3 cells were infected with
NS1-81 or NS1-156 virus, and the infected cells were labeled with
[35S]-Met-Cys as indicated above. The patterns of labeled
proteins obtained are shown in Fig. 2. As
expected, wild-type NS1 protein is absent (compare with Fig. 1A), and
mutant NS1-81 or NS1-156 proteins are expressed. Their identity was
confirmed by Western blot analysis with anti-NS1 serum (data not
shown). Despite lacking the C-terminal domain of NS1 protein, mutant
viruses NS1-81 and NS1-156 induced a shut-off similar to that observed
after wild-type influenza virus infection (compare with Fig. 1A), as
determined by the diminished labeling in the extracts obtained at late
times after infection and the disappearance of prominent cellular
bands, indicated with stars in Fig. 2A, whose quantification is
presented in Fig. 2B. Therefore, we can conclude that the interference
with cell pre-mRNA polyadenylation and nucleocytoplasmic transport induced by influenza virus infection plays no role in virus-induced shut-off. This is in line with the fact that most of the cell mRNAs
engaged in protein synthesis during the infection have been synthesized
and transported to the cytoplasm prior to the infection and would not
be affected by viral interference with nuclear events in gene
expression.
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FIG. 2.
Cell shut-off is not affected by C-terminal deletions of
NS1 protein. (A) Cultures of PKR+ or PKR NIH
3T3 cells were infected with the WSN mutant NS1-81 or NS1-156 at a
multiplicity of infection of 5 to 10 PFU per cell. The cultures were
labeled and processed as indicated in the legend to Fig. 1. The numbers
at the top of each lane indicate the time (in hours) after infection at
which the cultures were labeled. The main influenza virus proteins are
indicated to the right. The stars point to prominent cellular proteins
whose synthesis declined during the infection. (B) The bands indicated
with stars in panel A were quantified by microdensitometry. The results
have been normalized with respect to the initial pulse period.
|
|
In view of these results, we examined the integrity of the cellular
mRNAs present in the cytoplasm of cells infected with either
wild-type influenza virus or the NS1-81 mutant. Cultures of COS cells
were infected with either virus, and at various times after infection,
total cytoplasmic RNA was isolated and analyzed by Northern blotting
with an NP or a 
-tubulin cDNA probe, as previously described
(2). Along with the progressive accumulation of viral
mRNAs, as depicted by NP mRNA (Fig.
3A), the accumulation of -tubulin
mRNA was gradually decreased during the infection (Fig. 3A), both
in wild-type influenza virus-infected cells and in cells infected with
mutant NS1-81. This is in contrast to the high stability of -tubulin
mRNA in uninfected cells, measured by a classical actinomycin
D chase (2) (Fig. 3A). The quantification of these results
is presented in Fig. 3B and confirms previous reports that documented
the instability of cytoplasmic cellular mRNA after influenza
virus infection (2, 16). Furthermore, the results obtained
with NS1-81 mutant virus indicate that the C-terminal domain of NS1
protein does not play a role in such a phenomenon.
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FIG. 3.
Stability of cellular mRNA in cells infected with
wild-type (wt) or NS1-81 influenza viruses. (A) Cultures of COS-1 cells
were infected with either wild-type or NS1-81 viruses as indicated in
the legend to Fig. 1 or left uninfected. Some of the uninfected
cultures were treated with actinomycin D (100 µg/ml) (Act-D). At the
times indicated at the top of each lane, the cultures were fractionated
into nucleus and cytoplasm as described previously (25).
Total cytoplasmic RNA was extracted and analyzed by Northern
blotting with either NP or -tubulin probes labelled by
random priming of the corresponding cDNAs, as described
previously (2). (B) The intensity of the bands was
determined by microdensitometry and is represented as normalized with
respect to the uninfected cell extract (for -tubulin [-Tub]) or
the extract obtained at 8 h postinfection (for NP).
|
|
It has been proposed that influenza virus, like many other viruses, has
evolved mechanisms to avoid the activation of cellular PKR and hence a
total block of protein synthesis (19). These would include
the induction of expression of a cellular protein, p58, which inhibits
the autophosphorylation and activity of PKR (20), and the
expression of NS1 protein, a viral protein that has dsRNA-binding
activity (14) and would sequester dsRNA during the infection
(15, 21). In fact, it has been elegantly demonstrated that
NS1 protein plays an essential role in the anti-IFN response mediated
by influenza virus, because an NS1-null mutant virus can replicate in
cellular systems deficient in the IFN pathway, but not in normal cells
(11). Moreover, the anti-IFN action of NS1 protein is
directed to overcome the PKR response, since the NS1-null mutant virus
can replicate in PKR knockout mice (3). In addition to such
a blockade of PKR activation and/or activity, influenza virus takes
over the cellular protein synthesis machinery in such a way that at
late times in the infection, essentially only viral mRNAs are
translated. We have addressed the possible role of PKR activity in the
latter issue by single-cycle infections in normal versus PKR-defective
cells. Our results demonstrate that PKR activity is not relevant for
the preferential translation of viral mRNAs in the infection (Fig.
1). Furthermore, the use of C-terminally-deleted NS1 mutants indicates
that nuclear events in the cellular gene expression pathway do not play
a role in virus-induced shut-off (Fig. 2). Although it is clear that
cytoplasmic cellular mRNAs are degraded during the infection
(2, 16) (Fig. 3), the interpretation of these results is not
clear at present. In influenza virus-infected cells, viral mRNAs
are translated preferentially over cellular ones, a fact that may be
related to alterations in the cell translation apparatus (9)
and the activity of NS1 protein (6, 7) through its
association with the human homologue of Staufen protein (8,
23) and the eIF-4GI component of eIF-4F (1). It is
conceivable that such inhibition of cellular mRNA translation would
induce their destabilization, but we cannot exclude that virus
infection induces the selective degradation of cellular mRNAs and,
as a consequence, the inhibition of translation of cellular proteins.
| |
ACKNOWLEDGMENTS |
We are indebted to A. Nieto, D. Rodríguez, and A. Portela
for critical comments on the manuscript. We thank J. Gil, J. Pavlovic, and C. Weissmann for providing biological materials. The technical assistance of Y. Fernández and J. Fernández is gratefully acknowledged.
T. Zürcher was a fellow of the Swiss National Science Foundation.
R.M. Marión was a fellow of the Comunidad de Madrid. This work
was supported by Programa Sectorial de Promoción General del
Conocimiento (grant PB97-1160) and Comunidad de Madrid (grant 08.2/0025/98).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centro Nacional
de Biotecnologia (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain. Phone: 34 91 585 4557. Fax: 34 91 585 4506. E-mail:
jortin{at}cnb.uam.es.
Present address: GlaxoWellcome Medicines Research Centre,
Stevenage, Hertsfordshire SG1 2NY, United Kingdom.
| |
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Journal of Virology, September 2000, p. 8781-8784, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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