1 Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain
2 Center for Biosystems Research, University of Maryland Biotechnology Institute and VA-MD Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742, USA
Correspondence
Javier Benavente
bnjbena{at}usc.es
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ABSTRACT |
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INTRODUCTION |
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Avian reovirus genes are transcribed by a core-associated RNA polymerase to produce mRNAs that are identical to the positive strands of the dsRNA segments, possessing a type 1 cap at their 5' ends and lacking a 3' poly(A) tail (Martínez-Costas et al., 1995). Reoviral mRNAs are used as templates for the synthesis of viral proteins and minus-strand RNAs. Avian reovirus replication and morphogenesis occur within globular viral factories that are not microtubule-associated (Tourís-Otero et al., 2004a
).
The 367 aa avian reovirus NS protein, which is encoded by the S4 gene (Chiu & Lee, 1997
; Schnitzer, 1985
; Varela & Benavente, 1994
), binds single-stranded RNA (ssRNA) in a sequence-independent fashion (Yin & Lee, 1998
, 2000
) and is present in cytoplasmic globular inclusions in infected cells through an association with the non-structural protein µNS (Tourís-Otero et al., 2004b
). In this study, we have further characterized the nucleic acid-binding activity of avian reovirus
NS.
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METHODS |
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Cloning and generation of recombinant baculoviruses.
Cloning and sequencing of the recombinant plasmid pCR2.1-S4, containing the 1733 NS-encoding S4 gene, has been reported previously (Tourís-Otero et al., 2004b
). To express a recombinant
NS (r
NS) protein in insect cells, the S4-coding sequence of the pCR2.1-S4 plasmid was PCR-amplified by using the forward primer 5'-GGAATTCGCCATGGACAACACCGTGC-3' (EcoRI site underlined) and the reverse primer 5'-GCGTCTAGACTACGCCATCCTAGCTGG-3' (XbaI site underlined). The PCR product was digested and cloned into the EcoRI and XbaI sites of pFastBac1 (Bac-to-Bac system; Invitrogen) to generate pFastBac1-S4, which was then used to produce the baculovirus Bac-
NS according to the supplier's protocol. This baculovirus expresses the S4 gene under the control of the polyhedrin promoter.
To generate recombinant baculoviruses expressing deleted NS mutants, PCR amplification was performed with the following primers: to express the N-terminal mutant r
NS
N11, the forward primer was 5'-GGAATTCATGAACACATCCGGCGCACGTG-3' (EcoRI site underlined) and the reverse primer was 5'-GCGTCTAGACTACGCCATCCTAGCTGG-3' (XbaI site underlined). To express C-terminal mutants, the forward primer was 5'-GGAATTCGCCATGGACAACACCGTGC-3' and the reverse primers were 5'-GCGTCTAGATCAGATCATCCAATTACC-3' for
C16, 5'-GCGTCTAGACTAATTCATGGCGAAGCCCTG-3' for
C50 and 5'-GCGTCTAGACTACGCGTCCAACTCAACC-3' for
C100 (XbaI sites underlined). To express point
NS mutants, we performed site-directed mutagenesis of pFastBac1-S4 by using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Mutations were performed to change to leucine those lysine and arginine residues indicated in Table 1
. The mutant pFastBac1-S4 sequences were verified by restriction analysis and nucleotide sequencing. These mutants were used to generate recombinant baculoviruses as described above.
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For velocity-sedimentation analysis, S10 fractions were left untreated or treated with either 50 µg RNase A ml1 (Sigma-Aldrich) or 50 U RNase V1 ml1 (Ambion) for 15 min at 37 °C. An aliquot of the RNase-treated sample was adjusted to 1 M NaCl and incubated for 15 min at 37 °C. These samples, as well as a reticulocyte lysate that had been programmed with total RNA isolated from avian reovirus-infected cells, were loaded onto 1040 % sucrose gradients in STE buffer. After centrifugation at 150 000 g for 16 h at 4 °C in a Beckman SW50.1 rotor, 15 fractions of 300 µl were collected from the top of the tubes. These fractions and the pellets, as well as aliquots of both the original extract and purified avian reovirions, were boiled in Laemmli sample buffer and analysed by 12 % SDS-PAGE and autoradiography. Protein standards of known molecular masses and sedimentation coefficients were all from Sigma: BSA (66 kDa, 4·3S), gamma-globulin (156 kDa, 7S), catalase (250 kDa, 11·3S) and thyroglobulin (670 kDa, 19S). The standards were run on identical sucrose gradients, collected from the top and detected by measurement of A280.
For Sepharose bead-binding assays, the radioactive S10 fraction was subjected to ultracentrifugation (150 000 g for 2 h). Aliquots of the supernatant (100 µl) were supplemented with different NaCl concentrations and mixed with 50 µl of each of the various Sepharose beads indicated in Fig. 3 (Sigma). After incubation for 30 min at 4 °C in STE buffer, the mixtures were centrifuged for 30 s and subjected to five rounds of washing (with 1 ml STE buffer) and centrifugation. The pelleted beads were washed with STE buffer containing increasing salt concentrations (0·42·0 M NaCl) and centrifuged. The original extracts, the different washes and the final pellets were boiled in Laemmli sample buffer and analysed by 12 % SDS-PAGE and autoradiography. For competition assays, the supernatants of ultracentrifuged S10 extracts were incubated for 15 min at 4 °C in STE buffer with the indicated amounts of GTP or competitor soluble nucleic acids (0·5 mg ml1) prior to the binding assays.
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Immunoprecipitation, immunoblotting, chemical cross-linking and in vitro translation.
Immunoprecipitation and immunoblotting were performed as described previously (Tourís-Otero et al., 2004a). For chemical cross-linking, glutaraldehyde (Sigma) was diluted to the working concentration in PBS and incubated with purified r
NS for 30 min at room temperature. The reactions were stopped by adding Tris/HCl (pH 7·5) to a final concentration of 50 mM, followed by a 15 min incubation. Isolation of total RNA from avian reovirus-infected cells and its translation in rabbit reticulocyte lysates have been described previously (Varela & Benavente, 1994
).
Gel mobility-shift assays.
Uncapped and polyadenylated luciferase mRNA was purchased from Promega and poly(A) from Sigma. All other ssRNA probes were generated by in vitro transcription using the T7 RiboMAX Express RNA-production system (Promega). The DNA template for avian reovirus s1 mRNA transcription was generated by PCR amplification of S1 sequences contained within the pBsct-S1 plasmid (Bodelón et al., 2001) with the forward primer 5'-GCGTAATACGACTCACTATAGGCTTTTTCAATCCCTT-3' (T7 promoter sequence underlined) and the reverse primer 5'-GATGAATAACCAATCCCAGTAC-3'. The template for the synthesis of the 10 nt RNA was generated by annealing the oligonucleotides 5'-GCGTAATACGACTCACTATAGG-3' and 5'-GCGTGGTACCTATAGTGAGTCGTATTACGC-3'. The template for the synthesis of the 20 nt RNA was generated by annealing the oligonucleotides 5'-GCGTAATACGACTCACTATAGG-3' and 5'-GAGAATTCACGCGTGGTACCTATAGTGAGTCGTATTACGC-3'. The latter primer was also used as a 40 nt ssDNA probe. Annealing of this primer to its complement produced a 40 bp dsDNA probe. A 20 bp dsRNA was generated by annealing the 20 nt RNA with the RNA synthesized by T7 run-off transcription of NotI-predigested plasmid pCI-Neo.
The 5' end of the probes (20 pmol) was dephosphorylated by incubation with 20 U alkaline phosphatase (Roche Diagnostics) and then radiolabelled by incubation for 1 h at 37 °C with 50 µCi (1·85 MBq) [-32P]ATP (ICN) and 5 U T4 polynucleotide kinase (Promega) in 70 mM Tris/HCl (pH 7·6), 10 mM MgCl2 and 5 mM dithiothreitol. Unincorporated nucleotides were removed by Sepharose G50 chromatography.
Gel mobility-shift assays were performed by incubating 75 ng to 2 µg purified rNS in PBS with 10 000 c.p.m. of each radiolabelled probe in 20 µl STE buffer containing 10 U RNasin for 15 min at 4 °C. The samples were mixed with gel-loading buffer (0·25 % bromophenol blue, 0·25 % xylene cyanol, 30 % glycerol in H2O) and subjected to electrophoresis in 10 % polyacrylamide native gels in TBE buffer. At the end of the runs, the gels were dried and exposed to X-ray films. For competition assays, 1 µg purified r
NS was preincubated with 02 µg non-radioactive RNA prior to the addition of 50 ng radiolabelled s1 mRNA probe.
Membrane-filter assays.
North-Western blot assays were performed with extracts from insect cells that had been infected for 72 h with wild-type baculovirus or with recombinant baculovirus Bac-NS, as described by González & Ortín (1999)
. Proteins were separated in 12 % SDS-PAGE gels and transferred onto nitrocellulose membranes in 25 mM Tris, 192 mM glycine (pH 8·3). Membranes were incubated for 16 h at 4 °C in renaturing buffer [50 mM NaCl, 1 mM EDTA, 0·02 % Ficoll, 0·02 % BSA, 0·02 % polyvinylpyrrolidone, 0·1 % Triton X-100, 10 mM Tris/HCl (pH 7·5)], then incubated for 2 h at room temperature in renaturing buffer containing 106 c.p.m. 32P-radiolabelled s1 mRNA, washed four times (1 h each) with renaturing buffer and finally dried and exposed to X-ray film.
For membrane-filter assays, serial threefold dilutions of purified rNS, ranging from 0·1 to 9 µg, were prepared in STE buffer. BSA was then added to equalize the protein content of each sample to 9 µg and the final volume to 150 µl. Half of each sample was incubated with 3·5 M urea at 50 °C for 15 min and the other half was mock-incubated. Aliquots containing one-third of each sample were deposited onto three different 0·2 µm nitrocellulose membranes (Bio-Rad) by using a dot-blot apparatus (Millipore). The first membrane was incubated with Ponceau S solution (0·1 % Ponceau S in 5 % acetic acid; Sigma), the second membrane with anti-r
NS antibodies and the third membrane first for 1 h in STE buffer containing 0·5 mg BSA ml1 and then an additional hour in the same buffer containing 100 000 c.p.m. 32P-radiolabelled s1 mRNA. The membrane was finally washed five times in STE buffer containing 0·5 mg BSA ml1, then dried and exposed to X-ray film.
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RESULTS |
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Cross-linking with glutaraldehyde was performed next, to evaluate the oligomeric state of purified rNS. The immunoblot shown in Fig. 2(d)
revealed that, whereas the anti-
NS antibodies only recognized a 40 kDa protein band before cross-linking (lane 1), two additional bands of 80 and 110 kDa showed up after incubation with glutaraldehyde (lanes 2 and 3), suggesting that they correspond to
NS homodimers and homotrimers.
Binding to immobilized nucleic acids
To further characterize the in vitro nucleic acid-binding activity of NS, a cytoplasmic extract from 35S-amino acid-labelled avian reovirus-infected cells was ultracentrifuged and incubated with different resin-immobilized nucleic acids; the beads were subsequently washed with buffer containing increasing salt concentrations (Fig. 3a
). The radioactive proteins in the original extracts (lane 8), in the flow-through fractions (lane 1) and in the different washes (lanes 26), as well as those that remained attached to the matrix after the final wash (lane 7), were resolved by SDS-PAGE and visualized by autoradiography. Only the viral proteins
A and
NS displayed binding activity for the nucleic acids used in these assays, but not for control Sepharose beads (upper panel). Protein
A bound very tightly to poly(I : C), as it remained resin-bound even in the presence of 2 M NaCl and was able to bind poly(I : C)Sepharose in the presence of 2·5 M NaCl (Fig. 3b
). On the other hand,
NS bound with moderate affinity to poly(A), poly(U) and ssDNA, but not to poly(G), poly(C), poly(I : C) or dsDNA. Based on the salt concentration required for elution, we conclude that
NS exhibits a higher affinity for poly(A) than for poly(U), and for poly(U) than for ssDNA (Fig. 3a
). A similar conclusion was reached when the poly(A)-binding assays were performed with in vitro-translated
NS (data not shown), or when monitoring
NS attachment to, rather than elution from, the affinity beads at different salt concentrations (Fig. 3b
).
To further characterize the nucleic acid-binding activities of A and
NS, competition-binding assays with soluble, non-radioactive nucleic acids were performed. As shown in Fig. 3(c)
, the binding of
A to poly(I : C) could only be inhibited by soluble viral dsRNA and not by ssRNA, ssDNA or dsDNA, confirming previous observations that
A binds exclusively to dsRNA in a sequence-independent manner (Martínez-Costas et al., 2000
; Yin et al., 2000
). The binding of
NS to poly(A) and poly(U) could only be competed by soluble poly(A) or poly(U), not by the other nucleic acids tested.
It has been reported that GTP concentrations over 0·5 mM outcompete the binding of mammalian reovirus non-structural protein NS (mr
NS) to poly(A) and poly(U) (Richardson & Furuichi, 1985
). To assess whether a similar situation holds true for avian reovirus
NS (ar
NS), radiolabelled cytoplasmic extracts of both avian and mammalian reovirus-infected cells were supplemented with different GTP concentrations before incubation with poly(A)Sepharose beads. After washing the beads with binding buffer, the attached proteins were eluted by boiling in Laemmli sample buffer and analysed by SDS-PAGE and autoradiography (Fig. 3d
). Whilst GTP concentrations over 1 mM reduced the binding affinity of mr
NS to poly(A) (compare lanes 11 and 12), they apparently enhanced the affinity of ar
NS for poly(A) (compare lanes 5 and 6), suggesting that GTP has opposing effects on the RNA-binding activity of the two
NS proteins.
Gel mobility-shift assays
Consistent with the results of the Sepharose-immobilized nucleic acids, our gel-shift assays revealed that rNS forms complexes with radiolabelled ssRNA and ssDNA, but not with dsRNA or dsDNA (Fig. 4a
), and that the intensity of the ssRNAr
NS band was directly dependent upon the amount of r
NS used in the assays (Fig. 4b
). We further found that purified r
NS also forms complexes with poly(A) and luciferase mRNA (data not shown). When fixed amounts of r
NS were mixed with increasing amounts of unlabelled s1 mRNA or luciferase mRNA before the addition of the radiolabelled avian reovirus s1 mRNA probe, the two unlabelled mRNAs competed with similar efficiencies with radiolabelled s1 mRNA for r
NS binding (Fig. 4c
). A similar competition efficiency was also exhibited by poly(A) (data not shown). These results suggest that
NS does not have a preference for avian reovirus sequences.
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Filter-binding assays
The RNA-binding activity of rNS was also analysed by filter-binding assays. For this, the proteins present in extracts from both Bac-
NS- and wild-type baculovirus-infected Sf9 cells were resolved by SDS-PAGE and transferred onto nitrocellulose filters, and the filters were then probed with 32P-labelled s1 mRNA (Fig. 5a
). Whilst RNA binding by a cellular or baculovirus 30 kDa protein was evident in extracts from baculovirus-infected cells (lanes 3 and 4), no RNA binding by a protein migrating in the position of r
NS (40 kDa) could be observed (lane 4). These results suggest that filter-immobilized
NS monomers do not bind ssRNA. Further RNA-binding assays were performed with purified r
NS spotted directly onto membranes, either before or after denaturation with urea (Fig. 5b
). Ponceau S staining of one membrane revealed that all wells contained amounts of similar protein (top panel). Incubation of the second membrane with anti-
NS antibodies demonstrated that both native and denatured r
NS were retained by the membrane with similar efficiencies (medium panel). Probing the third membrane with 32P-labelled avian reovirus s1 mRNA revealed that only native r
NS displayed RNA-binding activity (bottom panel).
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We next generated recombinant baculoviruses that expressed proteins lacking the C-terminal 16, 50 and 100 residues from the C terminus of NS. Unfortunately, none of the C-terminally truncated proteins expressed in insect cells were found in the soluble fractions, which precluded their use for further analysis (Fig. 6b
). These results, however, suggest that the
NS C-terminal sequences are important for stability and/or solubility of the viral protein.
Site-directed mutagenesis was performed next to identify specific residues that are important for the activities of NS. An alignment of the avian reovirus 1733 and S1133
NS sequences with those of their counterparts from muscovy duck reovirus, Nelson Bay virus and mammalian reovirus type 3 revealed that the only conserved basic residues in all four proteins were those at avian reovirus
NS positions 6, 11, 29, 67, 80, 223, 234, 251, 262, 287, 310 and 365 (indicated by triangles in Fig. 7
). Recombinant baculoviruses expressing mutated
NS in which each of these basic residues was changed to leucines were generated and used to infect insect cells. All mutants were detected in the soluble fractions (Fig. 6c
), which allowed us to test their capacity to bind poly(A) and to form complexes. The results, summarized in Table 1
, revealed that mutation of the arginines at the
NS positions 6, 11, 234, 287 and 365 (indicated by filled triangles in Fig. 7
) prevented RNA binding and complex formation, whereas mutation of the other conserved basic residues did not (indicated by empty triangles in Fig. 7
). Furthermore, all mutants that did not bind RNA exhibited the same electrophoretic mobility as non-mutated r
NS, indicating that they did not undergo degradation (Fig. 6c
).
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DISCUSSION |
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Our results reveal that NS functions as an RNA-binding protein in infected cells and that it possesses ssRNA- and ssDNA-binding activity in vitro. Our observation that avian reovirus
NS binds poly(A) and poly(U), but not poly(C) or poly(G), suggests some specificity in the
NSRNA interaction. Although the significance of this specificity is not clear, it could be related to viral mRNA recognition, as all avian reovirus mRNAs sequenced so far contain 5'-GCUUUUU-3' and 5'-UUAUUCAUC-3' at their 5' and 3' ends, respectively, and these U-rich sequences are predicted to adopt a single-stranded conformation (data not shown). However, this hypothesis is not supported by our findings that r
NS does not have a preference for viral sequences in gel-shift assays and that r
NS associates with RNA in insect cells. Furthermore, the fact that poly(A), luciferase mRNA and s1 mRNA compete for the binding of
NS to the s1 mRNA with similar efficiencies suggests that
NS lacks sequence specificity and has no preference for capped and/or polyadenylated RNAs.
The possibility exists that viral cofactors enhance the RNA-binding specificity of NS within infected cells; a good candidate would be µNS, which associates with
NS and mediates its recruitment into viral factories (Tourís-Otero et al., 2004b
). Another possibility is that cellular mRNAs are excluded from the factories, obviating the need for inclusion-associated
NS to have a preference for viral sequences. In this scenario, sequence-independent RNA-binding activity would still allow
NS to concentrate and retain viral mRNAs within the factories, to provide them with RNase protection, to destabilize duplex regions and/or to improve their template efficiency for minus-strand synthesis. Sequestration of viral mRNAs within factories would direct them towards replication and would preclude their release for cytosolic translation.
The results of our RNA filter-binding assays suggest that native NS conformation is essential for RNA binding. One likely explanation is that
NS RNA-binding activity is dependent on multimerization, as has been reported to occur with rotavirus NSP2 (Boyle & Holmes, 1986
; Taraporewala et al., 1999
). Consistent with this hypothesis, native r
NS spotted onto a membrane filter binds RNA (Fig. 5b
, bottom panel), whereas the protein subjected to SDS-PAGE and renatured does not (Fig. 5a
, lane 4). These findings appear to suggest that oligomeric
NS, but not monomeric
NS, possesses RNA-binding activity. However, we cannot rule out the possibility that a properly folded
NS monomer binds RNA as well, and that the lack of RNA-binding activity shown by r
NS in the North-Western blot assay is due to inability of the renaturing conditions to induce correct
NS folding.
The fact that five conserved arginine residues, dispersed widely along the primary NS sequence, are important for poly(A) binding and complex formation suggests that
NS interacts with RNA through conformational domains and not through a linear RNA recognition motif, like the linear motifs found in various nucleic-acid binding proteins (Burd & Dreyfuss, 1994
; Kenan et al., 1991
; Kochan et al., 2003
; Lazinski et al., 1989
; Merrill et al., 1988
; Query et al., 1989
). One possibility is that
NS interacts with RNA through various domains located at different positions in the primary
NS sequence, as occurs with several other RNA-binding proteins (Cordingley et al., 1990
; Fillmore et al., 2002
; González & Ortín, 1999
; Rould et al., 1989
). Another possibility is that the five basic residues that are important for RNA binding may cluster together on native
NS to create a conformational RNA-binding motif. These basic residues are likely to be surface-exposed and to make direct contacts with RNA. Alternatively, they could be important for maintaining the native
NS conformation, although this possibility is unlikely, as the mutated r
NS versions that do not bind RNA still interact with µNS and are recruited to µNS inclusions (unpublished data). Furthermore, all point mutants and the N-terminal
NS truncation used in this study are present in the soluble fraction, whereas the
NS C-terminal truncations are insoluble. Collectively, these results suggest both that the C-terminal sequences of
NS are important for its conformational stability and that the point mutants and the N-terminal truncation are not misfolded.
The avian and mammalian reovirus NS proteins have similar characteristics. Both are proteins of
40 kDa that bind DNA and RNA non-specifically in vitro, that assemble into ribonucleoprotein complexes and that accumulate into viral factories through an association with µNS (Gillian & Nibert, 1998
; Gillian et al., 2000
; Gomatos et al., 1981
; Huismans & Joklik, 1976
; Miller et al., 2003
; Richardson & Furuichi, 1985
; Tourís-Otero et al., 2004b
; Yin & Lee, 1998
). However, the results of this study also revealed specific differences between the two proteins. Firstly, whereas the 4·37S RNA-free form of ar
NS exits as dimers and trimers, the RNA-free form of mr
NS appears to contain more monomeric subunits, as it sediments between 7 and 9 S (Gillian & Nibert, 1998
; Gillian et al., 2000
). Secondly, whilst mr
NS binds equally well to all four ribopolynucleotides at low salt concentrations and has a slight preference for poly(U) at 0·4 M NaCl (Huismans & Joklik, 1976
), ar
NS does not bind poly(C) or poly(G) and exhibits a slight preference for poly(A) over poly(U). Thirdly, GTP exerts opposite effects on the binding affinity of mr
NS and ar
NS to poly(A)Sepharose (Richardson & Furuichi, 1985
). Fourthly, mr
NS has been detected in the nucleus and cytoplasm of infected and transfected cells and has been reported to bind native and denatured DNA (Miller et al., 2003
; Shelton et al., 1981
), whereas ar
NS is not present within the nucleus and has no affinity for dsDNA (Tourís-Otero et al., 2004b
). Finally, the N-terminal 118 aa of mr
NS have been reported to be sufficient for RNA binding and complex formation (Gillian & Nibert, 1998
), whereas basic residues located at both the N and C termini of ar
NS are required for these activities.
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ACKNOWLEDGEMENTS |
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Received 30 July 2004;
accepted 11 January 2005.
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