| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Laboratoire de Virologie, CRSSA, BP 87, 38702 Grenoble, France
2 Unité de Génétique Moléculaire des Bunyaviridés, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
3 Laboratory of Virology, Wageningen University, The Netherlands
Correspondence
Michèle Bouloy
mbouloy{at}pasteur.fr
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Voinnet, 2005). The degradation is mediated by 21–24 nt long small interfering RNAs (siRNA), which are produced by the RNase III-like enzyme Dicer and incorporated into the RNA-induced silencing complex (RISC) (Elbashir et al., 2001; Hamilton & Baulcombe, 1999). The RNAi pathway is an ancient evolutionary conserved mechanism, which has been retained in plants, worms, insects and mammals and appears to represent an early form of innate immunity.
Among the different systems in which RNA silencing has been studied, plants, mammalian cells and insects (particularly Drosophila and mosquitoes) represent a significant field, but less is known about ticks and tick cells. Only a few reports described RNAi in ticks, in vitro or in vivo, and these studies describe silencing of specific genes but do not explore the mechanisms (Aljamali et al., 2003; Karim et al., 2004; Narasimhan et al., 2004). Ticks are obligate ectoparasites distributed worldwide and transmit human pathogens, such as the Lyme disease agent, the agent of Ehrlichiosis and arboviruses, some of which are serious human pathogens (i.e. the Crimean-Congo hemorrhagic fever virus). Recently, we showed that the tick cell line ISE6, established from Ixodes scapularis (Munderloh et al., 1994), was able to support the replication of the mosquito-borne Semliki Forest virus (SFV) and recombinant replicon (rSFV) and we found that infection of ISE6 cells with Hazara virus, a tick-borne arbovirus of the family Bunyaviridae (genus Nairovirus) was silenced through RNAi by a rSFV expressing nairovirus sequences (Garcia et al., 2005). The induction of RNAi in tick cells by SFV infection was clearly associated with the synthesis of siRNAs and a Dicer-like activity.
The discovery that plant viruses encode suppressors of gene silencing (Anandalakshmi et al., 1998; Beclin et al., 1998; Brigneti et al., 1998; Kasschau & Carrington, 1998; Vaucheret et al., 1998) provided a strong support that RNAi functions as a natural defence mechanism against viruses (Lindbo et al., 1993; Ratcliff et al., 1999). These inhibitors target various components of the RNAi pathway, thus representing valuable tools for the comprehension of the RNA silencing pathway. Among the best-studied suppressors, the helper component-proteinases (HC-Pro) encoded by potyviruses were shown to inhibit silencing of transgenes in transformed plants, antagonizing silencing in all tissues and targeting a step involved in the maintenance of silencing at or upstream from the production of siRNA (Anandalakshmi et al., 1998; Brigneti et al., 1998; Llave et al., 2000; Mallory et al., 2001). HC-Pro was shown to prevent dsRNA degradation into siRNAs (Bucher et al., 2003; Reavy et al., 2004). As RNA silencing is not limited to the plant kingdom, suppressors such as the B2 protein of the Flock house virus (Li et al., 2002; Lingel et al., 2005) have been found in insect and animal viruses and are able to act in cells from heterologous species. Among the inhibitors encoded by mammalian virus, NS1 of Influenza virus, which is acting as an interferon antagonist (Garcia-Sastre et al., 1998), exerts its suppressor activity through its dsRNA-binding domain, in plants, Drosophila and mosquito (Anopheles gambiae) cells (Bucher et al., 2004; Delgadillo et al., 2004; Li et al., 2004). Other non-structural proteins of negative-stranded or ambisense RNA viruses, NSs of Tospovirus Tomato spotted wilt virus (TSWV), NSs of La Crosse virus (genus Orthobunyavirus) and NS3 of Rice Hoja blanca virus (RHBV; genus Tenuivirus) were found to act as suppressors of post-transcriptional silencing of a transgene in mammals and plants (Bucher et al., 2003; Soldan et al., 2005). Interestingly, the NSs of TSWV seem to inhibit only the onset of sense transgene-induced post-transcriptional gene silencing (S-PTGS) but not inverted repeat PTGS (IR-PTGS) (Takeda et al., 2002).
To our knowledge, nothing is known of the ability of viral suppressors to abrogate RNAi in ticks and tick cells. In this work, we investigated whether suppressors of RNA silencing can act efficiently in tick cells and we set up a screening for RNAi inhibitors.
We chose to use a recombinant SFV to express heterelogous proteins and to induce RNAi in tick cells (Garcia et al., 2005). In the SFV replicon, the region encoding the viral structural proteins is replaced by the gene of interest. This replicon is complemented with a helper RNA that encodes the non-structural SFV proteins and allows production of recombinant SFV particles. This helper RNA lacks the encapsidation signal. The particles resulting from the coexpression of the replicon and helper RNAs cannot reinfect cells due to a mutation in the spike protein. They become infectious only when they are activated by 
-chymotrypsin (Salminen et al., 1992). During replication, the heterologous gene is expressed from the strong 26S promoter, but no viral particle is produced because the structural proteins are missing (Liljestrom & Garoff, 1991).
Since a key component in studies of RNAi suppressors is the system used to induce silencing, we tested the ability of a rSFV expressing firefly luciferase (SFV-Luc) to induce RNAi. SFV-Luc was prepared by inserting cDNA sequences amplified by PCR into the BamHI site of pSFV-1 plasmid (Liljestrom & Garoff, 1991). Recombinant plasmids were linearized at the unique SpeI site, were transcribed in vitro with SP6 RNA polymerase and electroporated into BHK-21 cells along with the Helper-2 RNA to produce suicide particles. We chose to express the firefly luciferase reporter protein because it could be examined in a simple assay and the half-life of the protein is relatively short (approx. 4 h), avoiding a steady-state effect. ISE6 cells were infected with SFV-Luc and luciferase activity from cells collected every day during 4 days post-infection (p.i.) was assayed using the Luciferase Assay System (Promega). Interestingly, this activity was highly expressed at 1 day p.i. but the level of expression decreased progressively from 2 to 4 days p.i. (Fig. 1a). Northern blots were carried out to analyse the viral genomic and 26S subgenomic RNAs. A significant decrease in SFV subgenomic mRNA was detected (data not shown), explaining reduction in protein expression.
|
). siRNAs migrating at the expected position with regard to the size markers were detected in cells infected with SFV-Luc (lane 1) but not in cells infected with SFV-1 or in the unprotected probe (lanes 2 and 3, respectively). Using a probe specific for the SFV replicon, siRNAs were detected in the SFV-Luc and SFV-1 infected cells (not shown), confirming that the replicon induces RNA silencing, whether it expresses a heterologous gene or not (Garcia et al., 2005). The RNase protection assay was chosen for its sensitivity but the frayed or overhanging ends produced by the RNase can give rise to siRNA heterogeneous in size (Sijen et al., 2001).
To investigate the ability to suppress RNAi in this system, we selected three different viral suppressors known as inhibitors in plants and/or mosquito cells: NS1 of influenza virus (NS1inf), NSs of TSWV (NSsTSWV) and HC-Pro of Zucchini yellow mosaic virus (ZYMV; HC-ProZYMV). The NS1 ORF of A/PR/8/34 strain and the TSWV NSs sequence were amplified from plasmids (Bucher et al., 2004), the HC-Pro ORF was derived from a plasmid containing ZYMV cDNA sequences, strain NAT [kindly provided by C. Desbiez, Institut National de la Recherche Agronomique (INRA), Montfavet, France and C. Antoniewski, Institut Pasteur, Paris, France]. The three rSFVs, SFV-NS1inf, SFV-NSsTSWV and SFV-HC-ProZYMV, were generated by inserting the specific cDNA in the SmaI site of the pSFV-1, after RT-PCR using oligonucleotides the sequences of which are available on request. Expression of these proteins in ISE6 cells infected with the corresponding rSFV was confirmed by indirect immunofluoresence assay using specific antisera (data not shown).
To evaluate the effect of the viral proteins on the suppression of RNAi in tick cells, we co-expressed the viral protein together with luciferase by co-infecting cells with SFV-Luc and SFV-NS1inf, SFV-NSsTSWV, SFV-HC-ProZYMV or SFV-1 as a control. Infections were carried out at an m.o.i. of 5 for each virus. The luciferase activity was determined at 3 days p.i., an incubation time when RNA silencing was effective in cells infected with SFV-Luc alone (see Fig. 1). Co-expression of NS1inf, NSsTSWV or HC-ProZYMV and firefly luciferase strongly increased the luciferase activity compared with the control in which SFV-1 was co-infected with SFV-Luc (Fig. 2a). Co-expression of SFV-NS1inf, SFV-NSsTSWV or SFV-HC-ProZYMV stimulated the level of luciferase activity by a factor of 4.5-, 2.5- and 1.5-fold (P<0.001, P<0.01, P<0.01, respectively, for the Student's t test), respectively. It should be noted that infection with SFV-Luc alone or co-infection with SFV-1 led to an almost identical level of reporter activity at this time, indicating a lack of suppressor activity from the SFV replicon. Additionally, the level of SFV-Luc subgenomic mRNA was clearly increased in cells expressing the suppressor (Fig. 2b). Altogether, the data indicated that the viral proteins identified as suppressors in plants and insect cells are also able to abrogate RNA silencing in tick cells.
|
), the NSs protein of Rift Valley fever virus (RVFV) was assayed for potential RNAi suppressive activity using the aforementioned tick-cell system. This experiment was carried out on the basis that the NSs of bunyaviruses share structural and functional similarities as they are encoded by the S segment and are involved in virulence (Blakqori & Weber, 2005; Bridgen et al., 2001). Notably, RVFV NS is a strong inhibitor of cellular transcription and an antagonist of interferon expression (Bouloy et al., 2001; Le May et al., 2004). Additionally, NSs of TSWV and RVFV, like the established RNA silencing suppressor protein NS3 of the tenuivirus RHBV (Bucher et al., 2003) are encoded by the same genomic organization using an ambisense strategy. However, no RNAi suppressive activity was observed in tick cells expressing RVFV NSs, confirming data obtained in plants and human 293T cells (E. Bucher, J. van der Velden, P. de Haan and M. Prins, unpublished). This suggests that the NSs proteins of bunyaviruses have evolved differently.
Viral suppressors have been described to abrogate RNA silencing at different steps of the RNAi pathway, some of them blocking the generation of the Dicer cleavage products, and others inhibiting a step further down. To determine at which step the viral suppressor acts, ISE6 cells were first infected with SFV-1 to induce the production of siRNAs and subsequently superinfected with SFV-Luc and rSFV expressing the suppressor. To validate the experimental conditions, we first verified that RNA silencing was established. When SFV-1 infected cells were superinfected with SFV-Luc at day 1, and reporter activity measured at day 4 p.i. (i.e. 3 days after superinfection), a strong reduction of reporter activity was observed in these cells (Fig. 3) compared with non-SFV-1-infected-SFV-Luc-superinfected cells. A reduction in the amount of subgenomic mRNA was similarly observed by Northern blot using a luciferase-specific probe (not shown). Together, these data clearly indicated that SFV-1 was the effector of RNA silencing. When cells infected with SFV-1 were superinfected with SFV-Luc and SFV-NS1, -NSs or -HC-Pro, the level of reporter activity clearly indicated that the presence of NS1 and, to some extent, of NSs partially, but significantly (Student's t test; P<0.01), restored the level of the reporter gene expression by a factor 3 and 1.5, respectively (Fig. 3). Interestingly, under these conditions and in contrast with the experiment described in Fig. 2, HC-Pro had no inhibitory effect on RNA silencing. The results obtained with HC-Pro are in accordance with published data showing that this protein acts at a maintenance step at or upstream of the production of siRNA (Llave et al., 2000; Mallory et al., 2001). For the NS1 protein, the partial inhibition of RNAi is associated with the protection of the luciferase mRNA against degradation (not shown), suggesting that NS1 inhibits RNAi in tick cells as in other cells, by sequestering the siRNAs through its RNA-binding activity (Bucher et al., 2004; Li et al., 2004).
|
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H. & Vance, V. B. (1998). A viral suppressor of gene silencing in plants. Proc Natl Acad Sci U S A 95, 13079–13084.
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, 313–317.[CrossRef][Medline]
Blakqori, G. & Weber, F. (2005). Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs. J Virol 79, 10420–10428.
Bouloy, M., Janzen, C., Vialat, P., Khun, H., Pavlovic, J., Huerre, M. & Haller, O. (2001). Genetic evidence for an interferon-antagonistic function of Rift Valley fever virus nonstructural protein NSs. J Virol 75, 1371–1377.
Bridgen, A., Weber, F., Fazakerley, J. K. & Elliott, R. M. (2001). Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis. Proc Natl Acad Sci U S A 98, 664–669.
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, 6739–6746.[CrossRef][Medline]
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, 1329–1336.[CrossRef][Medline]
Bucher, E., Hemmes, H., de Haan, P., Goldbach, R. & Prins, M. (2004). The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants. J Gen Virol 85, 983–991.
Delgadillo, M. O., Saenz, P., Salvador, B., Garcia, J. A. & Simon-Mateo, C. (2004). Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants. J Gen Virol 85, 993–999.
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, 494–498.[CrossRef][Medline]
Garcia, S., Billecocq, A., Crance, J. M., Munderloh, U., Garin, D. & Bouloy, M. (2005). Nairovirus RNA sequences expressed by a Semliki Forest virus replicon induce RNA interference in tick cells. J Virol 79, 8942–8947.
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, 324–330.[CrossRef][Medline]
Hamilton, A. J. & Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952.
Karim, S., Ramakrishnan, V. G., Tucker, J. S., Essenberg, R. C. & Sauer, J. R. (2004). Amblyomma americanum salivary gland homolog of nSec1 is essential for saliva protein secretion. Biochem Biophys Res Commun 324, 1256–1263.[CrossRef][Medline]
Kasschau, K. D. & Carrington, J. C. (1998). A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95, 461–470.[CrossRef][Medline]
Lecellier, C. H. & Voinnet, O. (2004). RNA silencing: no mercy for viruses? Immunol Rev 198, 285–303.[CrossRef][Medline]
Le May, N., Dubaele, S., Proietti De Santis, L., Billecocq, A., Bouloy, M. & Egly, J. M. (2004). TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus. Cell 116, 541–550.[CrossRef][Medline]
Li, H., Li, W. X. & Ding, S. W. (2002). Induction and suppression of RNA silencing by an animal virus. Science 296, 1319–1321.
Li, W. X., Li, H., Lu, R. & 9 other authors (2004). Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc Natl Acad Sci U S A 101, 1350–1355.
Liljestrom, P. & Garoff, H. (1991). A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology 9, 1356–1361.[CrossRef][Medline]
Lindbo, J. A., Silva-Rosales, L., Proebsting, W. M. & Dougherty, W. G. (1993). Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5, 1749–1759.[Abstract]
Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. (2005). The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double-stranded RNA recognition. EMBO Rep 6, 1149–1155.[CrossRef][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, 13401–13406.
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, 571–583.
Munderloh, U. G., Liu, Y., Wang, M., Chen, C. & Kurtti, T. J. (1994). Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. Formulation of medium for tick cell culture. J Parasitol 80, 533–543.[CrossRef][Medline]
Narasimhan, S., Montgomery, R. R., DePonte, K. & 7 other authors (2004). Disruption of Ixodes scapularis anticoagulation by using RNA interference. Proc Natl Acad Sci U S A 101, 1141–1146.
Ratcliff, F. G., MacFarlane, S. A. & Baulcombe, D. C. (1999). Gene silencing without DNA. RNA-mediated cross-protection between viruses. Plant Cell 11, 1207–1216.
Reavy, B., Dawson, S., Canto, T. & MacFarlane, S. A. (2004). Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells. BMC Biotechnol 4, 18.[CrossRef][Medline]
Salminen, A., Wahlberg, J. M., Lobigs, M., Liljestrom, P. & Garoff, H. (1992). Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry. J Cell Biol 116, 349–357.
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, 465–476.[CrossRef][Medline]
Soldan, S. S., Plassmeyer, M. L., Matukonis, M. K. & Gonzalez-Scarano, F. (2005). La Crosse virus nonstructural protein NSs counteracts the effects of short interfering RNA. J Virol 79, 234–244.
Takeda, A., Sugiyama, K., Nagano, H., Mori, M., Kaido, M., Mise, K., Tsuda, S. & Okuno, T. (2002). Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus. FEBS Lett 532, 75–79.[CrossRef][Medline]
Vaucheret, H., Beclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J. B., Mourrain, P., Palauqui, J. C. & Vernhettes, S. (1998). Transgene-induced gene silencing in plants. Plant J 16, 651–659.[CrossRef][Medline]
Voinnet, O. (2005). Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6, 206–220.[CrossRef][Medline]
Received 9 January 2006;
accepted 4 March 2006.
This article has been cited by other articles:
|
|
 |
|
  G. Blakqori, S. Delhaye, M. Habjan, C. D. Blair, I. Sanchez-Vargas, K. E. Olson, G. Attarzadeh-Yazdi, R. Fragkoudis, A. Kohl, U. Kalinke, et al. La Crosse Bunyavirus Nonstructural Protein NSs Serves To Suppress the Type I Interferon System of Mammalian Hosts J. Virol., May 15, 2007; 81(10): 4991 - 4999. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
