1 Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
2 Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, 28049 Madrid, Spain
Correspondence
Javier Benavente
bnjbena{at}usc.es
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ABSTRACT |
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Present address: Instituto de Biología Molecular Severo Ochoa, Campus Universidad Autónoma, 28049 Madrid, Spain.
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INTRODUCTION |
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IFN induces several different forms of 2-5A synthetase, which, on interaction with dsRNA, catalyse the conversion of ATP into short oligonucleotides of the general structure ppp(A2'p5')nA. These oligonucleotides bind and activate a latent endoribonuclease L (RNase L), which catalyses the indiscriminate degradation of RNAs, thereby leading to a general inhibition of intracellular protein synthesis (reviewed in Rebouillat & Hovanessian, 1999). RNase L has also been shown to cleave ribosomal RNAs (rRNAs) in a site-specific manner (Iordanov et al., 2000
; Rivas et al., 1998
; Wreschner et al., 1981
). On the other hand, activation of PKR by dsRNA interaction leads to phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2, giving rise to inhibition of protein synthesis by preventing the recycling of initiation factors (reviewed in Clemens, 1997
; Clemens & Elia, 1997
). Activation of these two IFN-induced antiviral systems has also been shown to trigger apoptosis in a way that is independent of the particular means of achieving translational inhibition (Iordanov et al., 2001
; Castelli et al., 1997
; Diaz-Guerra et al., 1997
; Lee & Esteban, 1994
).
Avian reoviruses are members of the Orthoreovirus genus, one of nine genera of the Reoviridae family. They are non-enveloped viruses that replicate in the cytoplasm of infected cells and contain ten dsRNA genome segments enclosed in a double protein capsid shell 7080 nm in diameter (reviewed in Robertson & Wilcox, 1986). The avian reovirus genome encodes at least ten structural proteins and four non-structural proteins, although very little is known about the functions of most of these proteins (Bodelon et al., 2001
; Varela et al., 1996
).
A previous study showed that four avian reovirus strains, including S1133, are resistant to the antiviral action of a natural chicken IFN produced in embryonated eggs (Ellis et al., 1983). A recent study, performed in our laboratory, revealed that exposure of chicken embryo fibroblasts (CEFs) to a recombinant chicken interferon (rcIFN) induces a strong intracellular antiviral state sufficient to inhibit the replication of vesicular stomatitis virus and vaccinia virus but not the replication of avian reovirus S1133. In the same study we also showed that the translation-inhibitory activity of dsRNA in reticulocyte lysates can be relieved by extracts of avian reovirus-infected cells, suggesting the presence of a protranslational factor in these extracts. Further in vitro translation experiments indirectly suggested that this factor is avian reovirus
A protein (Martínez-Costas et al., 2000
). Protein
A is a minor component of the virus inner capsid that binds dsRNA very tightly in a sequence-independent manner (Martinez-Costas et al., 1997
, 2000
; Yin et al., 2000
). However, no consensus dsRNA-binding motifs have been found in the amino acid sequence of
A protein, as found in the sequences of the dsRNA-binding proteins NS1 of influenza virus and VP6 of bluetongue virus (Hatada & Fukuda, 1992
; Stauber et al., 1997
).
In this study we have performed experiments to characterize the IFN-inhibitory activity of avian reovirus A protein. Our results suggest that
A is a key factor in the IFN-resistant phenotype displayed by avian reovirus because of its ability to down-regulate PKR function.
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METHODS |
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Plasmids.
To generate a plasmid expressing a maltose-binding protein (MBP)-tagged A fusion protein, total RNA from avian reovirus-infected cells was amplified by RT-PCR with the forward primer 5'-GCGGGATCCACGATGGCGCGTG-3' (BamHI site underlined) and the reverse primer 5'-GCGAAGCTTGCGTACGACCCTACGC-3' (HindIII site underlined). The resulting cDNA was digested and cloned into the BamHI and HindIII sites of pMalC (New England Biolabs) and the resulting recombinant plasmid, pMalCS2, was introduced into E. coli strain BL21. The correct orientation of the insert was confirmed by nucleotide sequencing.
Plasmid pPR15 (containing the luciferase reporter gene under the control of the vaccinia virus p4b late promoter), plasmid pPR35 (designed for IPTG-inducible expression of genes) and recombinant plasmid pPR35E3L have all been described previously (Diaz-Guerra et al., 1997; Rodriguez & Smith, 1990
). For the generation of the pPR35S2 plasmid and the recombinant vaccinia virus insertion plasmid pHLZ/S2, the S2 avian reovirus gene was excised from pMALCS2 plasmid by HindIII digestion and after filling the ends with Klenow DNA polymerase, the insert was digested again with BamHI. The resulting DNA fragment was then inserted into the BamHI and SmaI sites of the vaccinia virus insertion plasmid pPR35 and into the BglII and SmaI sites of the vaccinia virus insertion plasmid pHLZ (Rodriguez & Smith, 1990
).
Bacterial expression, protein purification and antibody generation.
For expression of MBP and MBPA, cultures of pMalC- and pMalCS2-transformed BL21 bacteria were grown in LB medium supplemented with 0·2 % glucose and 100 µg ampicillin ml-1 up to an optical density of 0·6 at 600 nm. The cells were then induced with 1 mM IPTG, incubated for 2 h at 37 °C and finally lysed by sonication in a buffer containing 0·25 % Tween 20, 1 mM DTT, 200 mM NaCl, 20 mM Tris/HCl, pH 7·5 and 1 mg lysozyme ml-1. The resulting extracts were clarified by centrifugation and MBP and MBP
A were purified from the supernatants using amyloseagarose columns, as described by the manufacturer (New England Biolabs). When indicated, protease factor Xa (New England Biolabs) was added to the MBP
A-containing sample at a w/w ratio of 1 % in column buffer (20 mM Tris/HCl, pH 7·4, 200 mM NaCl, 1 mM EDTA, 1 mM DTT) and incubated overnight at 4 °C. Protein
A was subsequently isolated from the mixture by Q-Sepharose chromatography performed in a buffer containing 20 mM Tris/HCl, pH 7·5, and 50 mM NaCl. Alternatively, supernatant extracts were subjected to poly(I : C)agarose chromatography (Amersham Bioscience), as previously described (Martínez-Costas et al., 2000
).
Preparation of rabbit polyclonal antibodies against both gel-purified and native A was carried out as described previously (Bodelon et al., 2001
).
Interferon treatment, infections, in vitro translation, protein analysis, detection of apoptosis and analysis of rRNA integrity.
Treatment of CEFs with rcIFN, infection of cell monolayers, in vitro translation, protein radiolabelling and SDS-PAGE analysis have all been described previously (Martínez-Costas et al., 2000). Densitometric analysis of viral protein bands was performed using a FluorS Multilmager system and Quantity One software (Bio-Rad).
Immunoprecipitation, immunoblotting and affinity chromatography methods have all been described previously (Bodelon et al., 2001; Martínez-Costas et al., 2000
). Detection of oligonucleosomal DNA fragments, estimation of the amount of cytoplasmic histone-associated DNA and analysis of the integrity of 18S and 28S rRNAs have also been described (Labrada et al., 2002
).
Transient transfection of HeLa cells.
Semiconfluent HeLa cell monolayers grown in 12-well plates were infected with 2·5 p.f.u. WRE3L- per cell and transfected 1 h later with 0·2 µg pPR15 per well plus the indicated amounts of pPR35-derived plasmids, using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Cells were then induced or not with 1·5 mM IPTG and lysed 24 h later. Luciferase activity in cell extracts was determined with a luminometer as previously described (Brasier et al., 1989).
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RESULTS AND DISCUSSION |
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Both native and gel-purified rA were used as immunogens for polyclonal antibody production. The proteins were subcutaneously injected into rabbits and 2 weeks after the second boost antisera were collected and tested. A subsequent Western blot analysis (Fig. 1C
) showed that while antiserum raised against gel-purified
A did not recognize any protein in extracts of either uninduced bacteria (Fig. 1C
, lane 1) or uninfected CEFs (Fig. 1C
, lane 3), it recognized both MBP
A in extracts of IPTG-induced pMalCS2-transformed bacteria (Fig. 1C
, lane 2) and naturally occurring
A protein present in extracts of avian reovirus-infected cells (Fig. 1C
, lane 4). On the other hand, while the antiserum raised against native r
A was able to immunoprecipitate MBP
A (Fig. 1D
, lane 2) and S1133
A (Fig. 1D
, lane 4) from 35S-labelled cell extracts and purified virions (Fig. 1D
, lanes 1 and 3), it did not immunoprecipitate any protein from extracts of either uninduced bacteria or uninfected CEFs (data not shown).
Protein A abolishes the inhibition of translation by dsRNA in vitro
The results of a previous study indirectly suggested that A protein is able to prevent the activation of the dsRNA-dependent enzymes in reticulocyte lysates (Martínez-Costas et al., 2000
). To obtain direct evidence for the protranslational activity of this protein, we compared the capability of MBP and MBP
A to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates (Fig. 2
). The results showed that, while MBP and MBP
A did not inhibit exogenous tobacco mosaic virus (TMV) mRNA translation (Fig. 2
, compare lanes 3 and 4 with lane 2), dsRNA induced a drastic inhibition of TMV protein synthesis (Fig. 2
, compare lane 5 with lane 2). Interestingly, whereas the inhibitory activity of dsRNA remained intact after preincubation with MBP (Fig. 2
, lane 6), it was completely abolished after preincubation with MBP
A (lane 7). This result indicates that
A is able to reverse the translation-inhibitory activity of dsRNA, probably because of its ability to sequester dsRNA from the dsRNA-dependent enzymes. To confirm this hypothesis, we next investigated how changes in the order of addition of dsRNA and MBP
A to reticulocyte lysates affected the translational efficiency of the reticulocyte lysate. Compared with the standard condition (Fig. 2
, lane 7), inhibition of translation was observed when the two compounds were added together without preincubation (Fig. 2
, lane 8) and this was even more pronounced when MBP
A was added 5 min later than dsRNA (Fig. 2
, lane 9). A similar translational rescue was also observed when dsRNA was preincubated with Xa-excised
A instead of MBP
A (results not shown).
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Transient expression of rA rescues WRE3L- gene expression in HeLa cells
As a first approach to document the capacity of rA to reverse the antiviral activity of IFN in vivo, we investigated the potential of the avian reovirus protein r
A to rescue the replication of the IFN-sensitive recombinant vaccinia virus WRE3L- in HeLa cells. This mutant virus lacks the E3L gene and in contrast to the wild-type vaccinia virus, its replication in HeLa cells is restricted and sensitive to IFN (Beattie et al., 1995
) and late in infection the mutant virus triggers apoptosis (Rivas et al., 1998
). To measure the ability of r
A to reverse blockage of WRE3L- in infected HeLa cells, we used a previously described transient transfectioninfection assay, which analyses the capability of proteins expressed from pPR35-derived plasmids to promote WRE3L- gene expression in HeLa cells (Rivas et al., 1998
). In this assay, promotion of WRE3L- gene expression is easily monitored by measuring the activity of luciferase expressed from plasmid pPR15, which contains the luciferase reporter gene under the control of the vaccinia virus late promoter p4b (Rodriguez & Smith, 1990
). The plasmid vectors used for cotransfection were the empty vaccinia virus insertional vector pPR35 and its derived vectors pPR35S2 and pPR35E3L, which contain the avian reovirus S2 gene and the vaccinia virus E3L gene, respectively, expressed from the late p4b promoter and controlled by two lac operator sequences. pPR35 plasmids also express the lac repressor from the constitutively active p7·5K vaccinia virus promoter.
HeLa cell monolayers were infected with 2·5 p.f.u. WRE3L- per cell and 1 h later cells were transfected with the luciferase reporter plasmid pPR15 plus one of the three vectors mentioned above. Infections were allowed to proceed for 24 h in the presence or absence of 1·5 mM IPTG, then cell extracts were prepared and luciferase activity was determined with a luminometer (Fig. 3). As expected, the levels of luciferase activity were low in uninduced cells and in IPTG-induced cells that had been transfected with the empty plasmid pPR35. However, luciferase levels augmented considerably after IPTG induction of cells transfected with the positive control plasmid pPR35E3L or the
A-encoding plasmid pPR35S2. These results strongly suggest that both avian reovirus
A protein and vaccinia virus E3L gene products are able to rescue WRE3L- gene expression in HeLa cells, probably because of their ability to bind dsRNA. Our finding that this increase is slightly higher in cells expressing
A than in those expressing E3L, together with the fact that E3L products have been shown to prevent activation of both PKR and 2-5A synthetase (Ho & Shuman, 1996
; Rivas et al., 1998
; Romano et al., 1998
), suggest that
A plays a critical role in modulation of the IFN-inducible antiviral enzymes.
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A comparative electrophoretic analysis of the 35S-labelled proteins synthesized in WR- and WRS2-infected CEFs (Fig. 4A) revealed that while there were no detectable differences in the protein pattern at the onset of the infection (Fig. 4A
, compare lanes 1 and 2), a prominent 40 kDa radioactive protein band was detected at 8 and 24 h post-infection (p.i.) in extracts of WRS2-infected cells (Fig. 4A
, lanes 4 and 6), but not in extracts of WR-infected cells (Fig. 4A
, lanes 3 and 5). The 40 kDa protein comigrated with the
A protein synthesized in avian reovirus-infected cells (Fig. 4A
, compare lanes 6 and 7). Immunoblot and immunoprecipitation analysis of cell extracts with polyclonal anti-
A antibodies confirmed the
A identity of the 40 kDa band present in WRS2-infected CEFs (Fig. 4B, C
). Affinity chromatographic assays on poly(I : C)agarose revealed that the r
A protein expressed by WRS2 displayed a dsRNA-binding affinity similar to that of the
A protein synthesized in avian reovirus-infected CEFs; the two proteins showed a very strong dsRNA-binding affinity, since they remained attached to the matrix after washing the dsRNAagarose beads with a buffer containing 2 M KCl (Fig. 4D
, lane 9 in panels WRS2 and S1133). Taken together, our results demonstrate that high levels of a functional
A protein are expressed by WRS2 in CEFs.
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Protein A protects vaccinia virus against the antiviral effects of IFN
To characterize the IFN-inhibitory activity of A protein, we next compared the effects of rcIFN on WRS2 and WRLuc replication in CEFs. WRLuc was chosen as a negative control virus because it expresses luciferase, which lacks IFN-inhibitory activity (Gherardi et al., 1999
). Plaque assay analysis of infectious progeny virus production (Fig. 5
A) revealed that WRS2 replication in CEFs is much more resistant to rcIFN than WRLuc replication, suggesting that
A protein confers vaccinia virus protection against IFN. A subsequent SDS-PAGE analysis of protein synthesis in rcIFN-treated cells (Fig. 5B
) showed that while the IFN treatment did not affect protein synthesis in uninfected cells (Fig. 5B
, compare lanes 1 and 2) and only induced a slight reduction of viral protein synthesis in WRS2-infected cells (Fig. 5B
, compare lanes 68), it caused a drastic inhibition of viral protein synthesis in WRLuc-infected cells (Fig. 5B
, compare lanes 35). A densitometric analysis of the two vaccinia virus protein bands marked with asterisks on the right of the autoradiogram shown in Fig. 5(B)
confirmed that viral protein synthesis in WRS2-infected CEFs was much more resistant to rcIFN than that in WRLuc-infected cells (Fig. 5C
). Taken together, these results suggest that IFN inhibits replication of vaccinia virus at a translational or a pretranslational step and that expression of avian reovirus
A protein relieves such inhibition.
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Since the above assays did not allow us to assess whether vaccinia virus susceptibility to IFN in CEFs is due to the activity of PKR and/or the 2-5A system, and since activation of enzymes of the 2-5A pathway has been shown to promote rRNA degradation into characteristic discrete fragments (Beattie et al., 1995; Iordanov et al., 2000
; Rivas et al., 1998
; Wrescher et al., 1981
), we next tried to assess the possible involvement of the 2-5A/RNase L pathway by examining the integrity of 28S and 18S rRNA in vaccinia virus-infected and interferon-treated CEFs. For this, total RNA isolated from cytoplasmic extracts of mock-infected and vaccinia virus-infected cells was stained with ethidium bromide, resolved in agarose/formaldehyde gels and subsequently visualized under UV light. This experimental approach has been successfully used in our laboratories to detect degradation of rRNAs in vaccinia virus-infected cells (Esteban et al., 1984
; Diaz-Guerra et al., 1997
). As can be seen in Fig. 6(B)
, the IFN treatment did not promote intracellular rRNA degradation in either uninfected or WRLuc- or WRS2-infected CEFs, confirming a previously published observation that ribosomal RNA is not degraded in IFN-treated vaccinia virus-infected CEFs (Grun et al., 1987
). These results suggest that RNase L is not active in IFN-treated vaccinia virus-infected chicken cells and therefore that the 2-5A system is not involved in the IFN-sensitive phenotype displayed by vaccinia virus in CEFs, despite the fact that chicken IFN induces very high intracellular levels of 2-5A synthetase in these cells (Martínez-Costas et al., 2000
; Ball & White, 1979
). This in turn would indicate both that PKR plays a key role in the IFN-sensitive phenotype displayed by vaccinia virus in CEFs and that the IFN/vaccinia virus/CEF system used in this study is a suitable system for directly testing the PKR-inhibitory activity of the product expressed by any gene inserted into the vaccinia virus genome.
Protein A exhibits PKR-inhibitory activity
Since A protects vaccinia virus against IFN in cells lacking RNase L activity, we next investigated whether this protection was due to interference with PKR activity. First, we evaluated the capacity of r
A to down-regulate the activity of a human recombinant PKR expressed by WR68K in BSC-40 cells. WR68K is an IPTG-inducible recombinant vaccinia virus that expresses a recombinant human PKR in BSC-40 cells (Lee & Esteban, 1993
; Lee et al., 1996
). On IPTG induction, the expressed kinase became intracellularly activated, leading to a strong inhibition of both intracellular translation and replication of the recombinant vaccinia virus (Lee & Esteban, 1993
; Fig. 7
A, compare lanes 1 and 2). As expected, IPTG-induction of BSC-40 cells coinfected with WR68K and WR or with WR68K and WRLuc caused a drastic reduction in both intracellular translation (Fig. 7A
, compare lanes 4 and 6 with lanes 3 and 5) and virus replication (data not shown), suggesting that PKR is not only expressed but also activated in these cells. In agreement with these findings, the intracellular levels of recombinant PKR did not increase significantly on IPTG induction (Fig. 7B
, lanes 16), probably because the translational block imposed by the active PKR inhibits WR68K gene expression and hence synthesis of the recombinant PKR encoded by the WR68K virus. In contrast, IPTG induction of cells coinfected with WR68K and WRS2 resulted in a much less pronounced reduction of protein synthesis (Fig. 7A
, compare lanes 7 and 8) and a considerable increase in the intracellular levels of recombinant PKR (Fig. 7B
, compare lanes 7 and 8), indicating that PKR kinase activity is dormant in these cells and that
A protein is able to rescue vaccinia virus replication by down-regulating PKR kinase activity.
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Several lines of evidence suggest that A protein down-regulates PKR function by preventing its activation, rather than by blocking its kinase activity: (i) in view of its strong dsRNA binding affinity,
A should prevent the activation of any dsRNA-dependent enzyme, as has been reported for other virus-encoded proteins (Gale & Katze, 1998
); (ii) our in vitro translation experiments demonstrate that
A inhibits the activation, not the activity, of endogenous dsRNA-dependent enzymes in reticulocyte lysates; (iii) transient expression of
A protein rescues E3L- vaccinia virus gene expression in HeLa cells, suggesting that both
A and E3L gene products use similar mechanisms for counteracting the antiviral effects of IFN; and (iv) it has recently been shown that expression of infectious bursal disease virus VP1/VP3 complexes in BSC-1 cells induces rRNA degradation because of the dsRNA polymerase activity of VP1 and that coexpression of the avian reovirus
A protein significantly reduces the rRNA degradation induced by VP1/VP3 complexes (A. Maraver, R. Clemente, J. F. Rodriguez & E. Lombardo, unpublished results) These data strongly suggest that
A is able to down-regulate PKR and the 2-5A synthetase/RNase L system in vivo and in vitro by sequestering dsRNA activators.
Like avian reoviruses, mammalian reoviruses also express a dsRNA-binding protein, the S4-encoded major outer capsid 3 protein, and several lines of evidence have indicated that protein
3 is likewise able to prevent PKR activation in vivo and in vitro (Bergeron et al., 1998
; Lloyd & Shatkin, 1992
; Yue & Shatkin, 1997
). However,
A possesses much stronger dsRNA-binding affinity than
3, since the former, but not the latter, remains attached to resin-coupled dsRNA at high salt concentrations (Martínez-Costas et al., 2000
; Yue & Shatkin, 1997
). Therefore, it would be expected that the efficiency of
A for sequestering dsRNA and preventing activation of the dsRNA-dependent enzymes is higher than that of
3, which might account for the higher IFN sensitivity of mammalian reoviruses in comparison with avian reoviruses (Martínez-Costas et al., 2000
; Jacobs & Ferguson, 1991
; Sherry et al., 1998
). This possibility is supported by the fact that the temperature-sensitive mammalian reovirus ts453 mutant, which expresses a
3 protein with increased dsRNA-binding affinity, is more resistant to IFN than wild-type virus (Bergeron et al., 1998
). On the other hand, the
A equivalent in mammalian reoviruses,
2 protein, has also been reported to be a dsRNA-binding protein, but it seems very unlikely that mammalian reovirus
2 plays a role in IFN resistance, since although it binds dsRNA in Northwestern blotting assays (Dermody et al., 1991
), it does not do so in assays performed in solution (our unpublished data). Thus, it seems that avian and mammalian reoviruses use different proteins for counteracting the antiviral action of IFN.
In conclusion, the results of the present study clearly demonstrate that A protein possesses IFN- and PKR-inhibitory activities and further suggest that
A plays a major role in the evasion of the antiviral activity of IFN in CEFs by avian reovirus by controlling the level of dsRNA in infected cells and hence blocking cellular response pathways dependent on dsRNA. Since no consensus dsRNA-binding sequences have been found in the primary structure of
A protein and since this protein displays especially tight binding to dsRNA, studies are currently under way to map the
A dsRNA-binding domain and to assess whether the IFN-inhibitory activity of
A protein relies exclusively on its ability to sequester dsRNA.
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ACKNOWLEDGEMENTS |
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Received 25 November 2002;
accepted 6 February 2003.
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