Journal of Virology, September 2000, p. 8781-8784, Vol. 74, No. 18
Centro Nacional de Biotecnología
(CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
Received 25 February 2000/Accepted 26 June 2000
The role of PKR activity in influenza virus-induced cell shut-off
was studied by infection of PKR+ or PKR 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 Cultures of NIH 3T3 cells (PKR+) or the corresponding cells
mutated at the pkr gene (PKR
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
ABSTRACT
Top
Abstract
Text
References
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
Top
Abstract
Text
References
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.
) (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.
View larger version (42K):
[in a new window]
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.
|
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.
|
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.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Aragón, T., S. de la Luna, I. Novoa, L. Carrasco, J. Ortín, and A. Nieto. Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus. Mol. Cell. Biol., in press. |
| 2. |
Beloso, A.,
C. Martínez,
J. Valcárcel,
J. Fernández-Santarén, and J. Ortín.
1992.
Degradation of cellular mRNA during influenza virus infection: its possible role in protein synthesis shutoff.
J. Gen. Virol.
73:575-581 |
| 3. |
Bergmann, M.,
A. Garcia-Sastre,
E. Carnero,
H. Pehamberger,
K. Wolff,
P. Palese, and T. Muster.
2000.
Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication.
J. Virol.
74:6203-6206 |
| 4. | Chen, Z., Y. Li, and R. M. Krug. 1999. Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery. EMBO J. 18:2273-2283[CrossRef][Medline]. |
| 5. | Clemens, M. J., and A. Elia. 1997. The double-stranded RNA-dependent protein kinase PKR: structure and function. J. Interferon Cytokine Res. 17:503-524[Medline]. |
| 6. | de la Luna, S., P. Fortes, A. Beloso, and J. Ortín. 1995. Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs. J. Virol. 69:2427-2433[Abstract]. |
| 7. |
Enami, K.,
T. A. Sato,
S. Nakada, and M. Enami.
1994.
Influenza virus NS1 protein stimulates translation of the M1 protein.
J. Virol.
68:1432-1437 |
| 8. |
Falcón, A. M.,
P. Fortes,
R. M. Marión,
A. Beloso, and J. Ortín.
1999.
Interaction of influenza virus NS1 protein and the human homologue of Staufen in vivo and in vitro.
Nucleic Acids Res.
27:2241-2247 |
| 9. |
Feigenblum, D., and R. J. Schneider.
1993.
Modification of eukaryotic initiation factor 4F during infection by influenza virus.
J. Virol.
67:3027-3035 |
| 10. | Gale, M. J., and M. G. Katze. 1998. Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol. Ther. 78:29-46[CrossRef][Medline]. |
| 11. | García-Sastre, A., A. Egorov, D. Matassov, S. Brandt, D. E. Levy, J. E. Durbin, P. Palese, and T. Muster. 1998. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252:324-330[CrossRef][Medline]. |
| 12. |
Garfinkel, M. S., and M. G. Katze.
1992.
Translational control by influenza virus. Selective and cap-dependent translation of viral mRNAs in infected cells.
J. Biol. Chem.
267:9383-9390 |
| 13. |
Garfinkel, M. S., and M. G. Katze.
1993.
Translational control by influenza virus. Selective translation is mediated by sequences within the viral mRNA 5'-untranslated region.
J. Biol. Chem.
268:22223-22226 |
| 14. |
Hatada, E., and R. Fukuda.
1992.
Binding of influenza A virus NS1 protein to dsRNA in vitro.
J. Gen. Virol.
73:3325-3329 |
| 15. |
Hatada, E.,
S. Saito, and R. Fukuda.
1999.
Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells.
J. Virol.
73:2425-2433 |
| 16. |
Inglis, S. C.
1982.
Inhibition of host protein synthesis and degradation of cellular mRNAs during infection by influenza and herpes simplex virus.
Mol. Cell. Biol.
2:1644-1648 |
| 17. |
Katze, M. G.,
D. DeCorato, and R. M. Krug.
1986.
Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus.
J. Virol.
60:1027-1039 |
| 18. |
Katze, M. G., and R. M. Krug.
1984.
Metabolism and expression of RNA polymerase II transcripts in influenza virus-infected cells.
Mol. Cell. Biol.
4:2198-2206 |
| 19. | Katze, M. G., and R. M. Krug. 1990. Translational control in influenza virus-infected cells. Enzyme 44:265-277[Medline]. |
| 20. |
Katze, M. G.,
J. Tomita,
T. Black,
R. M. Krug,
B. Safer, and A. Hovanessian.
1988.
Influenza virus regulates protein synthesis during infection by repressing autophosphorylation and activity of the cellular 68,000-Mr protein kinase.
J. Virol.
62:3710-3717 |
| 21. | Lu, Y., M. Wambach, M. G. Katze, and R. M. Krug. 1995. Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the eIF-2 translation initiation factor. Virology 214:222-228[CrossRef][Medline]. |
| 22. |
Marión, R. M.,
T. Aragón,
A. Beloso,
A. Nieto, and J. Ortín.
1997.
The N-terminal half of the influenza virus NS1 protein is sufficient for nuclear retention of mRNA and enhancement of viral mRNA translation.
Nucleic Acids Res.
25:4271-4277 |
| 23. |
Marión, R. M.,
P. Fortes,
A. Beloso,
C. Dotti, and J. Ortín.
1999.
A human sequence homologue of Staufen is an RNA-binding protein that is associated with polysomes and localizes to the rough endoplasmic reticulum.
Mol. Cell. Biol.
19:2212-2219 |
| 24. | Nemeroff, M. E., S. M. Barabino, Y. Li, W. Keller, and R. M. Krug. 1998. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3' end formation of cellular pre-mRNAs. Mol. Cell 1:991-1000[CrossRef][Medline]. |
| 25. |
Ortín, J., and W. Doerfler.
1975.
Transcription of the genome of adenovirus type 12. I. Viral mRNA in abortively infected and transformed cells.
J. Virol.
15:27-35 |
| 26. |
Park, Y. W., and M. G. Katze.
1995.
Translational control by influenza virus. Identification of cis-acting sequences and trans-acting factors which may regulate selective viral mRNA translation.
J. Biol. Chem.
270:28433-28439 |
| 27. | Shimizu, K., A. Iguchi, R. Gomyou, and Y. Ono. 1999. Influenza virus inhibits cleavage of the HSP70 pre-mRNAs at the polyadenylation site. Virology 254:213-219[CrossRef][Medline]. |
| 28. | Yang, Y. L., L. F. Reis, J. Pavlovic, A. Aguzzi, R. Schafer, A. Kumar, B. R. Williams, M. Aguet, and C. Weissmann. 1995. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J. 14:6095-6106[Medline]. |
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.
This article has been cited by other articles:
-
Rodriguez, A., Perez-Gonzalez, A., Hossain, M. J., Chen, L.-M., Rolling, T., Perez-Brena, P., Donis, R., Kochs, G., Nieto, A.
(2009). Attenuated Strains of Influenza A Viruses Do Not Induce Degradation of RNA Polymerase II. J. Virol.
83: 11166-11174
[Abstract] [Full Text] -
Richards, K. A., Chaves, F. A., Sant, A. J.
(2009). Infection of HLA-DR1 Transgenic Mice with a Human Isolate of Influenza A Virus (H1N1) Primes a Diverse CD4 T-Cell Repertoire That Includes CD4 T Cells with Heterosubtypic Cross-Reactivity to Avian (H5N1) Influenza Virus. J. Virol.
83: 6566-6577
[Abstract] [Full Text] -
Habjan, M., Pichlmair, A., Elliott, R. M., Overby, A. K., Glatter, T., Gstaiger, M., Superti-Furga, G., Unger, H., Weber, F.
(2009). NSs Protein of Rift Valley Fever Virus Induces the Specific Degradation of the Double-Stranded RNA-Dependent Protein Kinase. J. Virol.
83: 4365-4375
[Abstract] [Full Text] -
Garaigorta, U., Ortin, J.
(2007). Mutation analysis of a recombinant NS replicon shows that influenza virus NS1 protein blocks the splicing and nucleo-cytoplasmic transport of its own viral mRNA. Nucleic Acids Res
35: 4573-4582
[Abstract] [Full Text] -
Garcia, M. A., Gil, J., Ventoso, I., Guerra, S., Domingo, E., Rivas, C., Esteban, M.
(2006). Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action. Microbiol. Mol. Biol. Rev.
70: 1032-1060
[Abstract] [Full Text] -
Garaigorta, U., Falcon, A. M., Ortin, J.
(2005). Genetic Analysis of Influenza Virus NS1 Gene: a Temperature-Sensitive Mutant Shows Defective Formation of Virus Particles. J. Virol.
79: 15246-15257
[Abstract] [Full Text] -
Falcon, A. M., Marion, R. M., Zurcher, T., Gomez, P., Portela, A., Nieto, A., Ortin, J.
(2004). Defective RNA Replication and Late Gene Expression in Temperature-Sensitive Influenza Viruses Expressing Deleted Forms of the NS1 Protein. J. Virol.
78: 3880-3888
[Abstract] [Full Text] -
Burgui, I., Aragon, T., Ortin, J., Nieto, A.
(2003). PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes. J. Gen. Virol.
84: 3263-3274
[Abstract] [Full Text] -
Gastaminza, P., Perales, B., Falcon, A. M., Ortin, J.
(2003). Mutations in the N-Terminal Region of Influenza Virus PB2 Protein Affect Virus RNA Replication but Not Transcription. J. Virol.
77: 5098-5108
[Abstract] [Full Text] -
Salvatore, M., Basler, C. F., Parisien, J.-P., Horvath, C. M., Bourmakina, S., Zheng, H., Muster, T., Palese, P., Garcia-Sastre, A.
(2002). Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis. J. Virol.
76: 1206-1212
[Abstract] [Full Text] -
Ferko, B., Stasakova, J., Sereinig, S., Romanova, J., Katinger, D., Niebler, B., Katinger, H., Egorov, A.
(2001). Hyperattenuated Recombinant Influenza A Virus Nonstructural-Protein-Encoding Vectors Induce Human Immunodeficiency Virus Type 1 Nef-Specific Systemic and Mucosal Immune Responses in Mice. J. Virol.
75: 8899-8908
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»