1 Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
2 Viruvation BV, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
Correspondence
Marcel Prins
marcel.prins{at}wur.nl
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ABSTRACT |
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INTRODUCTION |
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Two functional classes of these molecules produced by DICER cleavage have thus far been identified: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The presence of these molecules is regarded as a hallmark of RNA silencing (Hamilton & Baulcombe, 1999). In plants, miRNAs seem to be predominantly involved in targeted mRNA degradation of transcription factors that play a role in development (Llave et al., 2002
; Palatnik et al., 2003
), while siRNAs recruit specific proteins to form the RNA-induced silencing complex (RISC) and initiate sequence-specific degradation of target RNAs, such as viral RNAs (reviewed by Vaucheret & Fagard, 2001
; Zamore, 2002
).
The siRNA-mediated RNA silencing machinery has been suggested to play different roles in different organisms. In plants, its major function seems to be providing antiviral defence at the nucleic acid level. Indeed, Arabidopsis mutants exhibiting impaired RNA silencing show enhanced susceptibility to virus infection (Dalmay et al., 2000; Mourrain et al., 2000
). In nematodes, it appears to stabilize the genome by inactivating transposable elements (Tabara et al., 1999
; Ketting et al., 1999
). In fission yeast, RNA silencing plays an important role in the regulation of the chromosome dynamics during cell division (Hall et al., 2003
). Interestingly, so far no naturally occurring siRNAs have been detected in mammalian cells, even though they are active in initiating RNA silencing when supplied in trans (McCaffrey et al., 2002
), a method generally used to silence endogenous genes (Elbashir et al., 2001
). Transfected siRNAs have been shown to protect mammalian cells efficiently against viral infection by providing sequence-specific intracellular immunity, suggesting that RNA silencing in mammalian cells can operate as an antiviral mechanism (Gitlin et al., 2002
).
To counteract the RNA silencing-based defence mechanism in plants, viruses encode specific proteins that have the ability to block various steps of the RNA silencing pathway (Brigneti et al., 1998; Voinnet et al., 1999
; Beclin et al., 1998
; Llave et al., 2000
). Many of these proteins have previously been linked to virulence of the virus. The HC-Pro protein of the plant-infecting potyviruses was one of the first such silencing suppressing proteins to be identified (Brigneti et al., 1998
). Suppression of RNA silencing by this protein is associated with a reduced accumulation of siRNAs, which is important for local RNA silencing (Llave et al., 2000
; Mallory et al., 2001
). Other viruses, such as Cucumber mosaic virus, express proteins that have the ability to stop the systemic silencing signal from spreading in the plant (Beclin et al., 1998
). Viral silencing suppressors have so far been identified in many positive-strand (Voinnet et al., 1999
; Brigneti et al., 1998
; Beclin et al., 1998
) and negative-strand RNA viruses (Bucher et al., 2003
) and DNA viruses (Voinnet et al., 1999
). The identification of RNA silencing suppressors has not remained limited to plant viruses, as the B2 protein of the insect-infecting Flock house virus has also been identified as a suppressor of RNA silencing, operating in plants as well as insect cells (Li et al., 2002
).
Recently, it was shown that the negative-strand RNA plant viruses Tomato spotted wilt virus (TSWV) and Rice Hoja blanca virus encode RNA silencing suppressors (Bucher et al., 2003). Like these two plant viruses, related vertebrate viruses also encode non-structural proteins with assigned virulence functions. Many of these proteins have been shown to interfere with innate defence responses of which the host interferon (IFN)-
/
-mediated response is the best studied (reviewed by Garcia-Sastre, 2001
).
One of the most extensively studied proteins with such a virulence function is the NS1 protein of influenza A virus (recently reviewed by Krug et al., 2003). For this multifunctional protein, three main functional domains have been proposed. On the N-terminal part of NS1 a domain involved in translational enhancement has been mapped between aa 81 and 113 (Aragon et al., 2000
). This domain has been shown to interact directly with the eukaryotic translation initiation factor 4GI (eIF4G) allowing preferential translation of the influenza virus messengers. The C-terminal half constitutes the effector domain, which has been reported to inhibit mRNA processing and nuclearcytoplasmic transport of host mRNAs (Lu et al., 1995
). It has been reported that NS1 interacts with a cleavage and polyadenylation specificity factor and the poly(A)-binding protein II required for 3'-end processing of cellular mRNAs (Chen et al., 1999
). This activity is thought to be required to prevent the maturation of host mRNAs encoding proteins with antiviral activity. Other reports suggest that the C-terminal region of the protein is mainly required for optimal dimerization of NS1 in vivo, which is required for its RNA-binding activity (Wang et al., 2002
). Additionally, NS1 contains a dsRNA-binding domain located at the N terminus of the protein (Hatada & Fukuda, 1992
; Wang et al., 1999
). This domain has been reported to be essential for its IFN-
/
antagonistic property (Wang et al., 2000
). It was suggested that the mechanism of the NS1 IFN-
/
antagonistic function could be achieved by sequestering dsRNA. This would result in preventing the activation of dsRNA-dependent protein kinase (PKR) and NF-
B and thereby in inhibiting the induction of IFN-
. Interestingly, sequestering of dsRNA molecules has also been described as the mode of action of one of the best-described plant viral RNA silencing suppressors in plants, p19 of the Tombusviruses, which directly associates with siRNAs (Silhavy et al., 2002
).
DsRNAs play a central role in RNA silencing as well as in the induction of the IFN pathway. Moreover, characteristics of the established RNA silencing suppressors of plant viruses correspond to some of the described properties of influenza virus NS1. Therefore, we wanted to investigate whether influenza A virus NS1 could play a role in suppressing RNA silencing, analogous to non-structural proteins of negative-strand plant viruses. Additionally, we tested whether this function of NS1 would be effectuated by sequestering siRNAs.
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METHODS |
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Isolation of siRNAs, Northern blotting and non-radioactive detection.
Transgenic leaf material was ground in liquid nitrogen and resuspended in 1·3 ml 2 % Sarkosyl/5 M NaCl (g leaf material)-1. After phenol extraction, polysaccharide contaminants were precipitated with 1 vol. 3 M ammonium acetate, pH 5·2 (Sharma et al., 2003). The water phase was ethanol precipitated and resuspended in TE. To remove larger RNA molecules, a PEG precipitation was performed using 5 % PEG 8000/0·5 M NaCl (final concentration) (Hamilton & Baulcombe, 1999
). The supernatant containing the siRNAs was precipitated with ethanol. Twenty µg total siRNAs per sample and 10 µg total RNA of the PEG 8000 precipitate were analysed by Northern blot using a GFP-specific DIG-labelled (Boehringer) PCR product to detect the GFP siRNAs and mRNAs, respectively.
Western blot analysis, NS1 and NS1rb protein production and purification, and siRNA-binding studies.
Total protein was extracted from infiltrated leaves and quantified using the Bradford assay (Bio-Rad). Ten µg total protein was analysed by Western blotting using anti-GFP and anti-rubisco (SSU) antibodies. The NS1 and NS1rb proteins were expressed in an N-terminally his-tagged form using the pQE31 vector system (Qiagen). The proteins were purified on TALON CellThru affinity columns (BD Biosiences). Synthetic double-stranded luciferase GL3 siRNAs were purchased from Qiagen. The 5'-phosphate groups of the siRNAs were removed by phosphatase treatment and replaced with 33P-radiolabelled phosphate groups. Plant siRNAs were enriched, radiolabelled and purified from an 8 % polyacrylamide gel using the radiolabelled luciferase siRNAs as markers. For the band-shift studies, various concentrations of NS1 or NS1rb protein were mixed with labelled siRNAs and incubated on ice for 20 min. A native 5 % polyacrylamide gel was used for sample analysis (Wang et al., 1999). Radiolabelled siRNAs were detected by autoradiography.
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RESULTS |
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When infiltrating Agrobacterium strains carrying the GFP reporter gene in combination with one expressing the NS1 gene product, highly enhanced GFP expression was observed (Fig. 1A). The level of GFP protein expression in NS1-expressing leaves was comparable to infiltrations with the established plant viral silencing suppressor NSS of TSWV and HC-Pro of the potyvirus Cowpea aphid-borne mosaic virus (CABMV) (Fig. 1B
). To verify that high GFP expression was indeed due to mRNA protection rather than an enhanced translation, Northern blot analyses were performed. The high accumulation of GFP mRNAs in plant leaves demonstrated that, as for TSWV NSS and CABMV HC-Pro, the NS1 protein protects the GFP mRNA from degradation by the RNA silencing machinery (Fig. 1C
).
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Expression of NS1 in a Potato virus X vector enhances its pathogenicity
In an extensive analysis, Brigneti and co-workers (1998) showed that the expression of viral silencing suppressing proteins in a Potato virus X (PVX) vector drastically increased symptom severity of the virus in N. benthamiana plants. To test the influence of NS1 on pathogenicity, we cloned the NS1 gene in both sense and anti-sense orientation into the pGR106 PVX vector, kindly provided by the Baulcombe group. Our GFP-silenced transgenic N. benthamiana line (Bucher et al., 2003) was then tooth-pick inoculated on a lower leaf with Agrobacterium containing pGR106 with NS1 in sense or anti-sense orientation and monitored for the development of viral symptoms. As shown in Fig. 2(A)
, the PVX vector expressing the NS1 protein (as confirmed by Western blot analysis, shown in Fig. 2
D) produced severe symptoms, while the anti-sense construct typically showed mild, wild-type-like symptoms (Fig. 2A
). To confirm reversal of the silenced state of the GFP transgene, systemically infected (top) leaves were monitored for GFP expression. The results showed that, on expression of NS1 from the PVX genome, local GFP-expressing patches could be observed (Fig. 2B, C
).
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The influenza virus NS1 protein binds siRNAs
NS1 has previously been reported to bind dsRNA molecules as short as 50140 nt (Wang et al., 1999). Combining the observation that NS1 is involved in RNA silencing suppression in plants with the fact that it requires a functional dsRNA-binding domain, we examined whether NS1 could exert this function by binding siRNAs.
For this purpose, gel-shift experiments were performed with purified NS1 protein produced in E. coli. As shown in Fig. 3(A), the NS1 protein was able to bind radiolabelled synthetic 21 bp siRNAs resulting in band shifting. Similarly, NS1 was capable of binding radiolabelled siRNAs extracted from plants (Fig. 3B
).
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DISCUSSION |
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We have demonstrated here that, as for established plant viral RNA silencing suppressors such as HC-Pro or NSS, the influenza A virus protein NS1 enhances expression of an Agrobacterium-delivered reporter protein construct by protecting its mRNAs from degradation. Concomitantly, siRNA production is drastically reduced, which indicates that NS1 interferes with the plant RNA silencing machinery. Additionally, here and in Delgadillo et al. (2004) (accompanying paper) it has been shown that expression of NS1 from a PVX viral vector results in strong enhancement of symptoms, a phenomenon often observed when expressing RNA silencing suppressors in plants (Brigneti et al., 1998
). Additionally, we have shown that the expression of NS1 in a PVX background leads to some local suppression of an established RNA silencing state in transgenic GFP-silenced plants.
The siRNA-binding capability of NS1 indicates that it may sequester free siRNAs during infection. By doing so it prevents the siRNAs from being incorporated into the RISC complex and taking part in the generation of longer dsRNA molecules and subsequent DICER/RISC-mediated degradative PCR of (viral) RNAs. In previous research, NS1 has been shown to bind longer dsRNAs (Wang et al., 1999), although it is less efficient in binding to dsRNAs than other cellular dsRNA-binding proteins (Krug et al., 2003
). Here we have shown that NS1 has a high affinity for siRNAs. It would be interesting to investigate whether NS1 has a higher affinity for the class of larger siRNAs (2426 nt), since independent research by Delgadillo et al. (2004)
shows that this size class is most affected by NS1 action. In plants, the smaller-sized class of siRNAs (2122 nt) has been implicated in suppression of local RNA silencing (RISC action), while the larger-sized class seems to be involved in systemic silencing and methylation status of homologous DNA (Hamilton et al., 2002
). As shown in Fig. 1
, NS1 has a clear effect on local RNA silencing leading to re-emergence of GFP expression, while Delgadillo et al. (2004)
have also shown an effect on the suppression of systemic silencing.
It can be argued that specific siRNA binding by NS1 merely reflects its general capacity to bind any dsRNA. Indeed, a recent report showed that several dsRNA-binding proteins were capable of acting as RNA silencing suppressors in plants (Lichner et al., 2003). It was suggested that they act by sequestering (larger) dsRNA molecules and hiding them from the silencing machinery. In addition to the NS1 protein, we have expressed the NSP5 protein of rotavirus, another viral dsRNA-binding protein (Vende et al., 2002
), in the Agrobacterium infiltration assay. However, expression of this protein did not result in RNA silencing suppression (results not shown) and may suggest that RNA silencing suppression is not a general feature of dsRNA-binding proteins.
Recent elegant work by the Burgyàn group has demonstrated that the P19 protein of the plant-infecting Tombusviruses suppresses RNA silencing in plants by sequestering siRNAs through direct binding (Silhavy et al., 2002). Here we have shown that, as well as its previously demonstrated activity of binding larger dsRNA molecules, NS1, like P19, efficiently binds siRNAs, either synthesized synthetically or isolated from plants. The dsRNA-binding motif in NS1 is required for binding siRNAs, as it is for its RNA silencing suppression activity in plants.
It may be assumed that, as in plants, NS1 binds siRNAs and longer dsRNAs in its natural host. In plants, the involvement of siRNAs is essential for virus inhibition through RNA silencing. In animals, larger dsRNA molecules (Williams, 1999) as well as siRNAs (Bridge et al., 2003
; Sledz et al., 2003
) have been demonstrated to be involved in the sequence-unspecific initiation of the IFN-mediated innate antiviral response. It has been demonstrated that dsRNA triggers the secretion of IFNs, which subsequently induce the production of PKR, 2'-5'-oligoadenylate synthetases and many more proteins with proposed antiviral activities (Rebouillat & Hovanessian, 1999
; Williams, 1999
; Kaufman, 1999
). Until recently, it was assumed that only longer stretches of dsRNA could induce the IFN response. However, recent reports suggest that short hairpin RNAs and siRNAs, designed to initiate RNA interference, can also trigger the IFN response (Bridge et al., 2003
; Sledz et al., 2003
).
As yet, it cannot be excluded that the function of NS1 in mammalian cells may be limited to sequestering siRNA and larger dsRNA molecules from detection by the IFN-/
response and PKR (Garcia-Sastre, 2001
). However, siRNA molecules, when transfected into mammalian cells, have been demonstrated to inhibit virus replication in a sequence-specific manner (Gitlin et al., 2002
; Andino, 2003
), suggesting an active sequence-specific RNA silencing machinery, which, as in plants and insects, can act as an antiviral response in mammalian cells. Taking this assumption further, this would imply that, next to or underlying the IFN-
/
response, RNA silencing may play a role in antiviral defence in mammals. It is interesting to note that the amino acids involved in the IFN antagonistic properties of NS1 in mammalian cells coincide with those essential for RNA silencing suppression in plants.
Taken together, we have demonstrated here that NS1 suppresses RNA silencing in plants by binding siRNAs. Our data thus suggest that RNA silencing may represent an important inhibitory function of virus infection that is counteracted by specific viral proteins, not only in plants, where it is well established, but also in animals. Clearly, to prove this final assumption, it will be of great importance to investigate the effects of NS1 on siRNA- and dsRNA-induced RNA silencing in mammalian systems using the recently established experimental protocols (Gitlin et al., 2002; McCaffrey et al., 2002
).
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ACKNOWLEDGEMENTS |
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NOTE ADDED IN PROOF |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aragon, T., de la Luna, S., Novoa, I., Carrasco, L., Ortin, J. & Nieto, A. (2000). Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus. Mol Cell Biol 20, 62596268.
Beclin, C., Berthome, R., Palauqui, J. C., Tepfer, M. & Vaucheret, H. (1998). Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes. Virology 252, 313317.[CrossRef][Medline]
Bergmann, M., Garcia-Sastre, A., Carnero, E., Pehamberger, H., Wolff, K., Palese, P. & Muster, T. (2000). Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. J Virol 74, 62036206.
Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363366.[CrossRef][Medline]
Bridge, A. J., Pebernard, S., Ducraux, A., Nicoulaz, A. & Iggo, R. (2003). Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 34, 263264.[CrossRef][Medline]
Brigneti, G., Voinnet, O., Li, W. X., Ji, L. H., Ding, S. W. & Baulcombe, D. C. (1998). Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 17, 67396746.
Bucher, E., Sijen, T., De Haan, P., Goldbach, R. & Prins, M. (2003). Negative-strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions. J Virol 77, 13291336.[CrossRef][Medline]
Chen, Z., Li, Y. & Krug, R. M. (1999). Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery. EMBO J 18, 22732283.
Cogoni, C. & Macino, G. (1997). Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc Natl Acad Sci U S A 94, 1023310238.
Dalmay, T., Hamilton, A., Rudd, S., Angell, S. & Baulcombe, D. C. (2000). An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543553.[Medline]
de Haan, P., Gielen, J. J. L., Prins, M., Wijkamp, I. G., Van Schepen, A., Peters, D., Van Grinsven, M. Q. J. M. & Goldach, R. (1992). Characterization of RNA-mediated resistance to tomato spotted wilt virus in transgenic plants. Bio/Technology 10, 11331137.[Medline]
Delgadillo, M. O., Sáenz, P., Salvador, B., García, J. A. & Simón-Mateo, C. (2004). Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants. J Gen Virol 85, 993999.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494498.[CrossRef][Medline]
English, J. J., Mueller, E. & Baulcombe, D. C. (1996). Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8, 179188.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806811.[CrossRef][Medline]
Garcia-Sastre, A. (2001). Inhibition of interferon-mediated antiviral responses by influenza A viruses and other negative-strand RNA viruses. Virology 279, 375384.[CrossRef][Medline]
Garcia-Sastre, A., Egorov, A., Matassov, D., Brandt, S., Levy, D. E., Durbin, J. E., Palese, P. & Muster, T. (1998). Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252, 324330.[CrossRef][Medline]
Gitlin, L., Karelsky, S. & Andino, R. (2002). Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418, 430434.[CrossRef][Medline]
Hall, I. M., Noma, K. & Grewal, S. I. (2003). RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast. Proc Natl Acad Sci U S A 100, 193198.
Hamilton, A. J. & Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950952.
Hamilton, A., Voinnet, O., Chappell, L. & Baulcombe, D. (2002). Two classes of short interfering RNA in RNA silencing. EMBO J 21, 46714679.
Hatada, E. & Fukuda, R. (1992). Binding of influenza A virus NS1 protein to dsRNA in vitro. J Gen Virol 73, 33253329.[Abstract]
Johansen, L. K. & Carrington, J. C. (2001). Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol 126, 930938.
Kaufman, R. J. (1999). Double-stranded RNA-activated protein kinase mediates virus-induced apoptosis: a new role for an old actor. Proc Natl Acad Sci U S A 96, 1169311695.
Ketting, R. F., Haverkamp, T. H., Van Luenen, H. G. & Plasterk, R. H. (1999). Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99, 133141.[Medline]
Krug, R. M., Yuan, W., Noah, D. L. & Latham, A. G. (2003). Intracellular warfare between human influenza viruses and human cells: the roles of the viral NS1 protein. Virology 309, 181189.[CrossRef][Medline]
Li, W. X. & Ding, S. W. (2001). Viral suppressors of RNA silencing. Curr Opin Biotechnol 12, 150154.[CrossRef][Medline]
Li, H., Li, W. X. & Ding, S. W. (2002). Induction and suppression of RNA silencing by an animal virus. Science 296, 13191321.
Lichner, Z., Silhavy, D. & Burgyan, J. (2003). Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences. J Gen Virol 84, 975980.
Lindbo, J. A. & Dougherty, W. G. (1992). Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts. Virology 189, 725733.[Medline]
Lipardi, C., Wei, Q. & Paterson, B. M. (2001). RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297307.[Medline]
Llave, C., Kasschau, K. D. & Carrington, J. C. (2000). Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci U S A 97, 1340113406.
Llave, C., Xie, Z., Kasschau, K. D. & Carrington, J. C. (2002). Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 20532056.
Lu, Y., Wambach, M., Katze, M. G. & Krug, R. M. (1995). Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. Virology 214, 222228.[CrossRef][Medline]
McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J. & Kay, M. A. (2002). RNA interference in adult mice. Nature 418, 3839.[CrossRef][Medline]
Mallory, A. C., Ely, L., Smith, T. H. & 7 other authors (2001). HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. Plant Cell 13, 571583.
Mourrain, P., Beclin, C., Elmayan, T. & 11 other authors (2000). Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101, 533542.[Medline]
Napoli, C. (1990). Introduction of chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279289.
Palatnik, J. F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J. C. & Weigel, D. (2003). Control of leaf morphogenesis by microRNAs. Nature 425, 257263.[CrossRef][Medline]
Qu, F. & Morris, T. J. (2002). Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing. Mol Plant Microbe Interact 15, 193202.[Medline]
Rebouillat, D. & Hovanessian, A. G. (1999). The human 2',5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties. J Interferon Cytokine Res 19, 295308.[CrossRef][Medline]
Sharma, A. D., Gill, P. K. & Singh, P. (2003). RNA isolation from plant tissues rich in polysaccharides. Anal Biochem 314, 319321.[CrossRef][Medline]
Sharp, P. A. (2001). RNA interference 2001. Genes Dev 15, 485490.
Sijen, T., Fleenor, J., Simmer, F., Thijssen, K. L., Parrish, S., Timmons, L., Plasterk, R. H. & Fire, A. (2001). On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107, 465476.[Medline]
Silhavy, D., Molnar, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M. & Burgyan, J. (2002). A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21, 30703080.
Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H. & Williams, B. R. (2003). Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834839.[CrossRef][Medline]
Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. & Mello, C. C. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123132.[Medline]
Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. & Sharp, P. A. (1999). Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13, 31913197.
Van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. & Stuitje, A. R. (1990). Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2, 291299.
Van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L. & 8 other (editors) (2000). Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press.
Vaucheret, H. & Fagard, M. (2001). Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet 17, 2935.[CrossRef][Medline]
Vende, P., Taraporewala, Z. F. & Patton, J. T. (2002). RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA. J Virol 76, 52915299.
Voinnet, O., Pinto, Y. M. & Baulcombe, D. C. (1999). Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96, 1414714152.
Voinnet, O., Lederer, C. & Baulcombe, D. C. (2000). A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157167.[Medline]
Wang, W., Riedel, K., Lynch, P., Chien, C. Y., Montelione, G. T. & Krug, R. M. (1999). RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. RNA 5, 195205.
Wang, X., Li, M., Zheng, H., Muster, T., Palese, P., Beg, A. A. & Garcia-Sastre, A. (2000). Influenza A virus NS1 protein prevents activation of NF-B and induction of alpha/beta interferon. J Virol 74, 1156611573.
Wang, X., Basler, C. F., Williams, B. R., Silverman, R. H., Palese, P. & Garcia-Sastre, A. (2002). Functional replacement of the carboxy-terminal two-thirds of the influenza A virus NS1 protein with short heterologous dimerization domains. J Virol 76, 1295112962.
Williams, B. R. (1999). PKR; a sentinel kinase for cellular stress. Oncogene 18, 61126120.[CrossRef][Medline]
Zamore, P. D. (2002). Ancient pathways programmed by small RNAs. Science 296, 12651269.
Received 20 October 2003;
accepted 2 December 2003.