1 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy
2 Department of Biología Molecular y Celular, Centro Nacional de Biotecnología, Cantoblanco, 28049 Madrid, Spain
Correspondence
Oscar R. Burrone
burrone{at}icgeb.org
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ABSTRACT |
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INTRODUCTION |
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The precise role of the non-structural proteins in viroplasms has not yet been fully characterized. NSP2 is a 35 kDa protein and has been described as a homo-octomer with nucleoside triphosphatase and helix-destabilizing activities (Schuck et al., 2001; Taraporewala et al., 2001
, 2002
; Taraporewala & Patton, 2001
; Jayaram et al., 2002
) and a possible role in unwinding of the viral dsRNA (Kattoura et al., 1992
; Taraporewala & Patton, 2001
). NSP5 comprises 198 amino acids with a high content of serines and threonines; it is O-glycosylated and highly phosphorylated (Welch et al., 1989
; Gonzalez & Burrone, 1991
; Afrikanova et al., 1996
; Blackhall et al., 1998
). The hyperphosphorylation produces isoforms with apparent molecular masses ranging from 28 to 3234 kDa, which can be easily visualized by SDS-PAGE. (Afrikanova et al., 1996
). It has recently been reported that NSP5 can bind dsRNA (Vende et al., 2002
). In addition, in viroplasms, NSP5 interacts with other viral proteins such as NSP2 (Poncet et al., 1997
; Afrikanova et al., 1998
), VP1 (Afrikanova et al., 1998
) and VP2 (Berois et al., 2003
). The in vivo interaction with NSP2 leads to NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs) (Afrikanova et al., 1998
; Fabbretti et al., 1999
). We have recently shown that the hyperphosphorylation is the consequence of a cellular kinase that is activated by NSP5 itself in a process resembling autophosphorylation (Eichwald et al., 2002
).
In this report, we present the mapping of the NSP2-binding regions in NSP5 and the time course of viroplasm formation post-infection.
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METHODS |
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To generate the recombinant rVV/NSP2 virus, BSC-40 cells were infected with recombinant vaccinia virus (rVV) VT7/LacOI (Ward et al., 1995) and transfected with pVOTE.1/NSP2. Selection and amplification were carried out as described by Earl & Moss (1993)
. The plasmid vector pVOTE.1 and VT7/LacOI were kindly provided by Bernard Moss (National Institutes of Health, Bethesda, MD).
Calcium phosphate transfection was performed essentially as described by Sambrook et al. (1989). Briefly, 1·5x106 cells were plated in 100 mm diameter dishes. Linearized plasmid DNA (6 µg) was resuspended in 50 µl 0·1x TE. Mix A was prepared by addition of 169 µl deionized water, followed by 5 µl 2 M CaCl2, 50 µl DNA, added drop by drop, and 26 µl 2 M CaCl2. This was then added to 250 µl mix B containing 2x HBS (280 mM NaCl, 10 mM KCl, 1·5 mM sodium phosphate, 12 mM glucose and 50 mM HEPES). The final mix was then added to the cells drop by drop. Cells were incubated overnight and the medium replaced with complete medium supplemented with 500 µg geneticin (G-418) ml-1 (Gibco-BRL, Life Technologies). Clones were picked after 1 week on selective medium.
Plasmid constructs.
An NSP2-encoding cDNA fragment, obtained by PCR amplification using specific primers to incorporate NcoI and BamHI restriction sites at the N and C termini, respectively, was cloned into pVOTE.1 (Ward et al., 1995) to obtain the construct pVOTE.1/NSP2. Constructs pT7v-NSP5, pT7v-
1, pT7v-
2, pT7v-
3, pT7v-
4, pT7v-
T, pT7v-
1/
2 and pT7v-
4T have been described previously (Afrikanova et al., 1998
; Fabbretti et al., 1999
; Eichwald et al., 2002
). The internal deletion plasmids pT7v-
2/
4T, pT7v-
2/
3 and pT7v-
3/
T were obtained by PCR using specific internal primers and cloned into KpnI and BamHI restriction sites in pcDNA3 (Invitrogen). Construct pEGFP-NSP5 has been previously described (Eichwald et al., 2002
), and pEGFP-NSP2 was obtained by insertion of the NSP2 fragment into pEGFP-N1 (Clontech) using KpnI and BamHI restriction sites. p(1-EGFP-4T), p(1-EGFP-T), p(1-EGFP) and p(EGFP-4T) were obtained by insertion of PCR-amplified fragments with EcoRI and BamHI restriction sites for domain 1 and BsrGI and NotI sites for domains 4T or T into pEGFP-N1. Constructs pT7v-(1-EGFP-4T), pT7v-(1-EGFP-T), pT7v-(1-EGFP) and pT7v-(EGFP-4T) were obtained by digestion from the constructs described above and cloned into pcDNA3 using restriction sites EcoRI and NotI. To produce yeast two-hybrid bait constructs, fragments corresponding to NSP5 mutants were obtained by PCR using specific primers to incorporate EcoRI and BamHI restriction sites at the N- and C-terminal ends, respectively. All these fragments were cloned into pBMT116 to obtain fusion constructs with LexA. NSP2 was cloned into the pVP16/D vector as a BssHIIXbaI fragment to generate the pVP16/NSP2 fusion construct (Visintin et al., 1999
).
Oligonucleotides.
The specific primers used for amplifying NSP2 were 5'-GATCCGTAGTCTAGAG-3' and 5'-TCGACTCTAGAGTACG-3'. The specific primers for amplifying pT7v-2/
4T were 5'-CGGGGTACCATGTCTCTCAGC-3' and 5'-CGCGGATCCTTAAGTTGAGATTGAT-3'; for pT7v-
3/
T, 5'-CGGGGTACCATGTCTCTCAGC-3' and 5'-CGCGGATCCTTAGTACTTTTTCTTA-3'; and for pT7v-
2/
3, 5'-CGGGGTACCATGTCTCTCAGC-3' and 5'-GCGGGATCCTTACAAATCTTCGATC-3'. The specific primers used for amplifying p(1-EGFP-4T), p(1-EGFP-T), p(1-EGFP) and p(EGFP-4T) were 5'-CCGGAATTCATGTCTCTCAGCATTG-3' and 5'-CGCGGATCCGCAGATTTTCCAGA-3' for region 1; and 5'-CGGTGTACATTGATAATAAAGAG-3' and 5'-TAAAGCGGCCGCTTACAAATCTTCGATC-3' for region 4T. The tail (T) was obtained by annealing of the oligonucleotides 5'-GTACATTGCACTAAGAATGAGGATGAAGCAAGTCGCAATGCAATTGATCGAAGATTTGTAAGC-3' and 5'-GGCCGCTTACAAATCTTCGATCAATTGCATTGCGACTTGCTTCATCCTCATTCTTAGTGCAAT-3'. The specific primers used for amplification of pBMT-NSP5, pBMT-
2, pBMT-
3, pBMT-
4 and pBMT-
2/
3 were 5'-CCGGAATTCATGTCTCTCAGCATTG-3' and 5'-GCGGGATCCTTACAAATCTTCGATC-3'; for pBMT-
1 and pBMT-
1/
3, 5'-CGGGAATTCATGATTGGTAGGAG-3' and 5'-GCGGGATCCTTACAAATCTTCGATC-3'; and for pBMT-
C48, 5'-CCGGAATTCATGTCTCTCAGCATTG-3' and 5'-TGATCAGCGAGCTCTAGC-3'.
Localization to viroplasms and VLS formation.
Transfection, infection with rotavirus and immunofluorescence for visualization of viroplasms were performed as described (Eichwald et al., 2002). For VLS formation, cells were infected with rVV/NSP2 at a multiplicity of 3 p.f.u. per cell. After 1 h, cells were transfected with 2 µg plasmid and induced with 1 mM IPTG and 100 µg rifampicin ml-1 (Sigma) to prevent vaccinia virus morphogenesis (Grimley et al., 1970
; Nagaya et al., 1970
). At 18 h post-infection, cells were fixed in 3·7 % paraformaldehyde and immunofluorescence was performed as described previously (Eichwald et al., 2002
).
Quantification of viroplasms.
Cells were analysed using a confocal microscope (Axiovert; Zeiss). The area of the viroplasm was measured with the LSM 510 software version 2.02, using the overlay option that allows the measurement of functions such as length, angle, area and circumference. The analysis was performed on 20 cells per experiment and the results were plotted with the Microsoft Excel 9.0 program.
Yeast two-hybrid system.
The yeast growth and the two-hybrid system were carried out as described previously (Visintin et al., 1999). Briefly, plasmids were transformed into the L40 yeast strain using the lithium acetate transformation protocol (Gietz et al., 1992
). Positive clones were selected using auxotrophic markers for both plasmids (Trp and Leu) and prototrophy markers Uracil (U), Lys (K) and His (H). Yeast colonies grown on plates lacking histidine were obtained after 3 days of culture at 30 °C. For
-galactosidase assays, clones were lysed in liquid nitrogen and assayed for
-galactosidase activity as described previously (Breeden & Nasmyth, 1985
). Positive clones developed an intense blue colour after 15 min, while negative clones had not developed a blue colour after 12 h.
In vivo binding/immunoprecipitation assay.
MA104 cells (0·5x106 cells) were infected with rVV/NSP2 at a multiplicity of 3 p.f.u. cell-1. After 1 h, cells were transfected with 2 µg of each NSP5 deletion mutant in 5 µl Transfectam (Promega) and induced with 1 mM IPTG. Four hours later, cells were incubated in methionine-free DMEM for 30 min. The medium was then replaced with DMEM containing 1·5 mg methionine l-1 and 1 mM IPTG, 100 µCi [35S]methionine was added and the cells were labelled for 18 h. Before lysis, cells were washed twice with PBS, incubated for 10 min in 25 mM DSP (dithiobis-succinimidyl propionate; Pierce) in PBS at 4 °C, then washed three times in 2·5 ml 50 mM Tris/HCl, pH 7·5, 150 mM NaCl. Cells were lysed in 60 µl TNN lysis buffer [100 mM Tris/HCl, pH 8·0, 250 mM NaCl, 0·5 % NP-40, 1x protease inhibitor cocktail (Sigma)] for 10 min at 4 °C and centrifuged at 10 000 g for 5 min. Supernatants were immunoprecipitated as described previously (Eichwald et al., 2002). Beads were washed twice in TNN and once in RIPA (100 mM Tris/HCl, pH 8, 150 mM NaCl, 1 % NP-40, 0·5 % deoxycholate, 0·1 % SDS) and the samples were analysed by SDS-PAGE (Laemmli, 1970
). Visualization of 35S-labelled proteins was enhanced by fluorography using Amplify (Amersham). Autoradiography was performed at -70 °C using X-ray film (Kodak X-OMAT AR). Quantification of labelled proteins was carried out by densitometry on a Pharmacia LKB-Ultrascan XL densitometer and the values corrected according to the number of methionines in each protein. The ratio value was calculated using the formula: ratio=[a*(b/c)] day-1, where a=densitometric value of the NSP5 deletion mutant, b=number of methionines in the full-length NSP5, c=number of methionines in the deletion mutant NSP5 and d=densitometric value of NSP2. The mean was calculated from three independent experiments for each deletion mutant. The relative binding of full-length NSP5 was defined as 1.
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RESULTS |
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Binding assay in mammalian cells
As an alternative method for studying NSP5NSP2 interactions, we performed a binding/immunoprecipitation assay from total extracts of cells expressing NSP2 and various NSP5 mutants. For this purpose, the NSP2 gene was subcloned into the vaccinia virus insertion/expression vector pVOTE.1 and used to generate an IPTG-inducible recombinant vaccinia virus for NSP2 (rVV/NSP2) (Ward et al., 1995). As shown in Fig. 5
(A), expression of NSP2 was obtained from IPTG-induced MA104 cells infected with rVV/NSP2 and metabolically labelled with [35S]methionine. Following immunoprecipitation with anti-NSP2 serum, a single band of approximately 35 kDa with mobility identical to NSP2 from rotavirus strains OSU and SA11 was obtained.
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A representative binding assay is shown in Fig. 5(B) for some of the mutants. As expected, no cross-reactivity was observed in immunoprecipitations with anti-NSP5 in cells that were only infected with rVV/NSP2 and expressed no NSP5 (lane 1). In Fig. 5(C)
, the relative binding of the different mutants is shown with respect to wtNSP5, which was defined as having a value of 1. Each NSP5 deletion mutant experiment was performed three times and the arithmetic mean was calculated. Deletion of region 1 (mutants
1,
1/
2 and
1/
3) had a profound effect on the ability to bind NSP2. Similarly, deletion of the C-terminal region T (mutants
T,
4T,
2/
4T and
3/
T) also resulted in strong binding impairment. These results were in agreement with the data obtained in the yeast two-hybrid experiments and indicated that the N and C termini are relevant for NSP2 binding. Independent deletion of other regions (such as regions 2, 3 and 4) suggested that they were not directly involved. Interestingly, region 3 appeared to have an inhibitory effect on the ability of the wild-type protein to bind NSP2, since deletion mutant
3 showed a relative binding twofold higher than wtNSP5. However, when regions 1 or T from this mutant were also deleted (
1/
3 or
3/
T), NSP2 binding was completely abolished.
Viroplasm localization is dependent on regions 1 and T
To demonstrate further that the N- and C-terminal regions of NSP5 are indeed the only ones required for interaction with NSP2 and localization to viroplasms, we turned to new constructs in which region 1 was fused to the N terminus of EGFP, and regions 4, T or both to the C terminus (Fig. 6). The different constructs were used to study the formation of VLSs by co-transfection with NSP2 and localization in the viroplasm of virus-infected cells. VLSs were only obtained with mutants containing both region 1 and T, while localization to viroplasms could be seen even when region 1 was not present, i.e. in EGFP-4T. This is likely to be the consequence of EGFP-4T interaction with viral NSP5, which depends on NSP5 C-terminal residues (Torres-Vega et al., 2000
; Eichwald et al., 2002
).
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DISCUSSION |
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Viroplasms appear to be dynamic structures, with an increase in size and a reduction in number over time, suggesting a fusion process between them. Moreover, the stack images suggested that viroplasms are well-defined spherical structures. In reoviruses, it has also been reported that NS, the non-structural protein homologous to rotavirus NSP2 with ssRNA binding activity and capacity to form higher-order homo-oligomeric structures (Huismans & Joklik, 1976
; Richardson & Furuichi, 1985
; Gillian & Nibert, 1998
; Gillian et al., 2000
), can also form inclusion-body-like structures when co-expressed with reovirus protein µNS (Becker et al., 2003
). NS2 of bluetongue virus and rotavirus NSP2 have been considered to be proteins of similar function, since they share NTPase activity, non-specific ssRNA binding and localization to inclusions bodies (Taraporewala et al., 2001
; Fillmore et al., 2002
). However, there are no reports describing the ability of NS2 to form VLSs when co-expressed with other viral proteins. With regard to rotavirus NSP5, no analogous function has been reported for any other viral protein of viruses in the Reoviridae family.
A main goal of our study was to map the binding sites of NSP5 for NSP2. For this purpose we used two different in vivo strategies: (i) a yeast two-hybrid system; and (ii) an in vivo binding/immunoprecipitation assay in mammalian cells. Essentially, the two methods gave similar results, indicating the N- and C-terminal parts of NSP5 as the main components required for the interaction. The results of the yeast two-hybrid system suggested that the central region of 30 amino acids (region 3) could also be important for NSP2 binding. However, the direct assay of the mutant lacking only region 3 (3) was not possible because the construct LexA
3 was transactivating in the absence of NSP2VP16. This was also the case for wtNSP5 and mutant
1. In the immunoprecipitation assay, however, deletion of region 3 produced an increased NSP2 binding activity, suggesting not only that it is not involved in binding, but also that it behaves as an inhibitory domain. On the other hand, the relevant roles of regions 1 and 4T were clearly confirmed. In addition, these two terminal regions were able to confer to EGFP, when fused at the N and C terminus, respectively, the ability to localize to viroplasms in virus-infected cells and to form VLSs in cells expressing NSP2. The fact that the construct containing only the C terminal fusion 4T from NSP5 was unable to form VLSs suggested that region 1 indeed plays a crucial role in NSP2 binding, as has been previously proposed (Eichwald et al., 2002
).
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ACKNOWLEDGEMENTS |
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Received 29 August 2003;
accepted 13 November 2003.