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International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, 34012 Trieste, Italy
Correspondence
Oscar R. Burrone
burrone{at}icgeb.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Institut Curie, INSERM U520, 12 rue Lhomond, 75005 Paris, France. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Chen et al., 1990; Gonzalez & Burrone, 1991; Kattoura et al., 1992, 1994; Afrikanova et al., 1996, 1998; Aponte et al., 1996; Poncet et al., 1997; Taraporewala et al., 1999; Taraporewala & Patton, 2001; Eichwald et al., 2002; Berois et al., 2003). We have recently reported that NSP5 is also essential for viroplasm assembly (Vascotto et al., 2004). In some group A rotavirus strains, such as SA11, the genome segment 11 (gs11) also encodes a second non-structural protein, termed NSP6, of 91 residues, from a different ORF (Mattion et al., 1991). In the porcine OSU strain, however, this second ORF encodes a truncated protein of 51 amino acids (ORF2) (Gonzalez & Burrone, 1989). Due in part to the lack of a reverse genetics system in rotavirus, no clear function in the viral replicative cycle has yet been found for NSP5. Here, we used the RNA interference strategy directed towards gs11 mRNA to demonstrate that NSP5 is an essential protein for the formation of viroplasms and for virus replication.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Eichwald et al., 2004). SA11 and OSU rotaviruses were propagated in MA104 cells as described by Estes et al. (1979). Virus titres were determined by immunofluorescence on MA104 cells with anti-NSP2 antibody or by direct determination of viroplasms in the NSP2-EGFP stable cell line.
Small interfering (si) RNAs and transfection.
The siRNAs were chemically synthesized with a 3' TT deoxynucleotide overhang on both strands and obtained as annealed duplexes (IBA). A fluoresceinated siRNA against SA11 segment 11 (si/SA11-fluo) with 6-FAM on the 5' end of the sense strand was obtained as an annealed duplex (Qiagen). Approximately 1·5x105 cells were transfected with 2 µg siRNAs in 1 ml serum-free medium containing 5 µl Transfectam reagent (Promega). After 6 h at 37 °C, cells were washed twice with serum-free medium and incubated for an additional 66–72 h in medium supplemented with 10 % fetal bovine serum (Gibco). This time point was found to be the optimum for the interfering effect (Dector et al., 2002). All experiments with siRNA were repeated several times and the data shown are representative of the results obtained.
Western immunoblot analysis.
Cells were lysed and Western blots performed as described previously (Eichwald et al., 2002). SDS-PAGE (Laemmli, 1970) was done with equal amounts of proteins. Membranes were incubated with guinea pig sera against viral proteins NSP5, NSP2, VP7 and VP1, followed by goat anti-guinea pig HRP-conjugated antibody (KPL), and developed with the ECL system (Amersham). Antibodies for NSP5 and NSP2 were prepared as described by Eichwald et al. (2002). Anti-VP7 and anti-VP1 sera were obtained by immunization of guinea pigs with glutathione S-transferase- and His-tagged fusion proteins, respectively, essentially as described by Gonzalez & Burrone (1991).
Immunofluorescence.
Immunofluorescence was performed essentially as described previously (Eichwald et al., 2002). Slides were mounted with ProLong mounting medium (Molecular Probes) and acquired with cool SNAPs system using a fluorescence microscope (DMLB; Leica). Around 500 cells were counted per experimental point and the experiment was repeated at least three times. Viroplasm-positive cells were considered those with two or more dots per cell.
Viral genomic dsRNA.
Equal numbers of rotavirus-infected cells were collected and total genomic dsRNA was prepared as described previously (Chen et al., 1990). Samples were electrophoresed in a 10 % polyacrylamide gel, 0·75 mm thick, for 15 h at 20 mA and bands were visualized with ethidium bromide.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The two synthetic siRNAs (termed si/SA11 and si/OSU) contain in each strand a 3' end with a TT deoxynucleotide overhang (Fig. 1b). To test the activity of the siRNAs, we transfected them into MA104 cells, which were infected 72 h later with both rotavirus strains. When NSP5 expression was analysed at 5 h post-infection, a large reduction in the amount of SA11 or OSU NSP5 was observed in cells transfected with the corresponding siRNA, while practically no effect was observed in cells infected with either virus that had received the non-homologous siRNA (Fig 1c). At longer times post-infection (15 h), a significantly larger amount of NSP5 was observed, probably due to cells that did not uptake the siRNA or to the fact that continuous production of viral mRNA overrides the interfering activity (Figure 1d). These results indicated that both siRNAs were highly efficient in specifically targeting the homologous gs11 mRNA, producing NSP5 protein-knockout phenotypes. We also carried out Western-blot analysis for other viral proteins that do (VP1, NSP2) and do not (VP7) localize in viroplasms. A strong inhibition of the accumulation of all three viral proteins took place concomitantly with the reduction of NSP5, while this was not the case for the control cellular protein actin (Fig. 1c).
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. The effect was highly strain specific: si/SA11 and si/OSU strongly inhibited the emergence of viroplasms in cells infected, respectively, with SA11 (75–80 % inhibition) and OSU (80–83 % inhibition), while cells infected with the non-homologous strains were fully competent in sustaining viroplasm assembly. Moreover, the few viroplasms remaining in siRNA-treated cells appeared smaller and reduced in number. This was due to the lack of NSP5, and not NSP6, since OSU-NSP5 was able to complement viroplasms formation in si/SA11-treated cells infected with SA11 virus. This was shown using C7 cells, an MA104 stable transfectant expressing NSP5-OSU which is sensitive to si/OSU but not to si/SA11 (data not shown). These cells showed a significant increase, compared with MA104 cells, in the emergence of viroplasms when infected with SA11 following si/SA11 treatment (Fig. 2a). As expected, the same C7 cells did not rescue viroplasm formation when infected with OSU following si/OSU treatment (not shown). Using a fluoresceinated si/SA11 (si/SA11-fluo), we estimated that more than 80 % of cells were transfected and unable to form viroplasms after SA11 infection, while infection with OSU was unaffected (Fig. 2b). The fluorescent si/SA11 was found as punctate foci in the cytoplasm adjacent to the nucleus, a localization characteristic of siRNAs (Kapadia et al., 2003).
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), suggesting impaired virus replication. To investigate this aspect further, we analysed production of infective virions and of viral dsRNA in SA11-infected or OSU-infected cells. Production of infective virions by siRNA-treated cells was visualized directly by infecting a stable transfectant cell line expressing NSP2–EGFP fusion protein, which shows, upon virus infection, relocalization to viroplasms of the cytoplasmic diffused protein (Eichwald et al., 2004). Alternatively, the same virus inocula were used to infect MA104 cells and the appearance of viroplasms analysed by immunofluorescence with an anti-NSP2 serum.
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, si/SA11, but not si/OSU, inhibited the production of infective SA11 virus. Analogous results were obtained with OSU infections in si/SA11- or si/OSU-treated cells (not shown). Virus titres were determined in cells infected with either virus strain and treated with the different siRNAs. As shown in Table 1, virus titres were reduced between 10- and 20-fold depending on the virus strain in cells treated with the corresponding homologous siRNA. It is worth noting that, since not all cells can be transfected with siRNA, a number of them will sustain virus replication. In different experiments, we observed that the reduction in OSU titres was always higher than that obtained with SA11, consistent with what we observed by immunofluorescence.
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). Furthermore, as for the formation of viroplasms, complementation of SA11 dsRNA production was observed in si/SA11-treated C7 cells that express OSU-NSP5 but not for OSU replication following si/OSU treatment. This result also indicated that NSP6, as it is the case for OSU, is not essential for SA11 replication.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kojima et al., 1996) while, in the porcine OSU strain, a premature termination codon reduces the coding region to 51 residues (Gonzalez & Burrone, 1989). Since it is not yet possible to generate recombinant rotaviruses for a reverse genetic approach to study the function of viral proteins, we followed the RNA interference strategy. To knock down gs11 mRNA of two different rotavirus strains (simian SA11 and porcine OSU), a stretch of 19 nucleotides with a single nucleotide difference between the two strains was targeted with siRNAs specific for each of them. Both siRNAs were entirely specific, abolishing expression of NSP5 only for the virus encoding the fully homologous mRNA. This fact provided important internal controls for the siRNAs effects, ruling out a general alteration of siRNA-treated cells, since they were able to sustain replication of the viral strain with the non-homologous gs11 mRNA to a level comparable to that of control, untreated cells.
We found a strong inhibition of viroplasm formation in siRNA-treated, virus-infected cells. This result was in complete agreement with our recent finding on the inhibition of viroplasm assembly by specific anti-NSP5 intrabodies (Vascotto et al., 2004). Moreover, in line with the intrabody protein-knockout strategy, we also demonstrated that NSP5 and not NSP6 is the relevant protein for virus replication. Complementation was achieved with the NSP5 gene encoding a truncated version of NSP6 (OSU-NSP5). The phenotype of virus infection in NSP5-depleted cells resembled that of an NSP2 ts mutant, which was viroplasm- and replication-negative at the non-permissive temperature (Ramig & Petrie, 1984; Chen et al., 1990), as well as the phenotype of cells treated with RNA interference for NSP2 (Silvestri et al., 2004). Furthermore, the reduction of virus titres that we obtained was similar to what was found by RNA interference of NSP2 (Silvestri et al., 2004). These are important findings considering the close interaction of NSP5 and NSP2 in viroplasms, as well as their ability to associate to form viroplasm-like particles when co-expressed in the absence of virus replication (Patton & Gallegos, 1988; Gallegos & Patton, 1989; Fabbretti et al., 1999). The data suggest that both NSP5 and NSP2 play structurally relevant roles in the assembly of viroplasms.
The large reduction in the amount of other viral proteins observed in NSP5-depleted (siRNA-treated) cells suggested that most of the viral proteins found in virus-infected cells are, rather than the product of the internalized infecting virions, the consequence of the translation of mRNAs produced by de novo-formed particles (secondary transcription) that have not yet completed the final maturation steps, as has been shown for reovirus (Zweerink & Joklik, 1970; Acs et al., 1971; Sakuma & Watanabe, 1971; Skup & Millward, 1980). This interpretation is consistent with the active transcription of viral genes occurring in double-layered particles derived from purified mature infective virions (Bican et al., 1982; Kohli et al., 1993; Patton & Chen, 1999; Lawton et al., 2000; Thouvenin et al., 2001) and with the results of RNA interference for NSP2 (Silvestri et al., 2004). However, it has recently been suggested that subvirus replication intermediates, rather than double-layered particles, are responsible for secondary transcription (Lopez et al., 2005).
In conclusion, the data presented demonstrated that NSP5 is an essential protein for the formation of viroplasms and for virus replication and suggested that this non-structural protein can be considered a good target for therapeutic purposes. However, it remains to be determined whether NSP5, in addition to its crucial role in shaping the architecture of viroplasm, also participates directly in other steps of virus morphogenesis. The RNA interference approach, in combination with complementation assays with defined mutants, opens new opportunities to understand the function of viral proteins in the absence of a reverse genetic system.
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ACKNOWLEDGEMENTS |
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Received 9 September 2004;
accepted 23 December 2004.
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