Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation

Catherine Eichwald1, José Francisco Rodriguez2 and Oscar R. Burrone1

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


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viroplasms are discrete structures formed in the cytoplasm of rotavirus-infected cells and constitute the replication machinery of the virus. The non-structural proteins NSP2 and NSP5 localize in viroplasms together with other viral proteins, including the polymerase VP1, VP3 and the main inner-core protein, VP2. NSP2 and NSP5 interact with each other, activating NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs). We have used NSP2 and NSP5 fused to the enhanced green fluorescent protein (EGFP) to investigate the localization of both proteins within viroplasms in virus-infected cells, as well as the dynamics of viroplasm formation. The number of viroplasms was shown first to increase and then to decrease with time post-infection, while the area of each one increased, suggesting the occurrence of fusions. The interaction between NSP2 and a series of NSP5 mutants was investigated using two different assays, a yeast two-hybrid system and an in vivo binding/immunoprecipitation assay. Both methods gave comparable results, indicating that the N-terminal region (33 aa) as well as the C-terminal part (aa 131–198) of NSP5 are required for binding to NSP2. When fused to the N and C terminus of EGFP, respectively, these two regions were able to confer the ability to localize in the viroplasm and to form VLSs with NSP2.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
After entering the cell, rotaviruses initiate active transcription of viral genes to produce viral mRNAs. Within 2–3 h post-infection, discrete cytoplasmic structures called viroplasms are formed, which contain the non-structural proteins NSP2 and NSP5, together with other viral proteins, including the polymerase, VP1, the guanyltransferase methylase, VP3, and the main inner-core protein, VP2 (Gallegos & Patton, 1989; Zeng et al., 1998). It is in the viroplasm where the initial steps of viral morphogenesis occur, in a process involving the concerted packaging and replication of the 11 positive-polarity ssRNAs to generate the viral dsRNA genomic segments (Patton & Gallegos, 1990; Wentz et al., 1996; Patton et al., 1997).

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 32–34 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.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
MA104 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % foetal calf serum (Life Technologies). BSC-40 cells were cultured in DMEM supplemented with 10 % newborn calf serum (Hyclone). Rotavirus simian strain SA11 (serotype 3) and porcine strain OSU (serotype 5) were propagated and grown in MA104 cells as previously described (Estes et al., 1979).

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-{Delta}1, pT7v-{Delta}2, pT7v-{Delta}3, pT7v-{Delta}4, pT7v-{Delta}T, pT7v-{Delta}1/{Delta}2 and pT7v-{Delta}4T have been described previously (Afrikanova et al., 1998; Fabbretti et al., 1999; Eichwald et al., 2002). The internal deletion plasmids pT7v-{Delta}2/{Delta}4T, pT7v-{Delta}2/{Delta}3 and pT7v-{Delta}3/{Delta}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 BssHII–XbaI 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-{Delta}2/{Delta}4T were 5'-CGGGGTACCATGTCTCTCAGC-3' and 5'-CGCGGATCCTTAAGTTGAGATTGAT-3'; for pT7v-{Delta}3/{Delta}T, 5'-CGGGGTACCATGTCTCTCAGC-3' and 5'-CGCGGATCCTTAGTACTTTTTCTTA-3'; and for pT7v-{Delta}2/{Delta}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-{Delta}2, pBMT-{Delta}3, pBMT-{Delta}4 and pBMT-{Delta}2/{Delta}3 were 5'-CCGGAATTCATGTCTCTCAGCATTG-3' and 5'-GCGGGATCCTTACAAATCTTCGATC-3'; for pBMT-{Delta}1 and pBMT-{Delta}1/{Delta}3, 5'-CGGGAATTCATGATTGGTAGGAG-3' and 5'-GCGGGATCCTTACAAATCTTCGATC-3'; and for pBMT-{Delta}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 {beta}-galactosidase assays, clones were lysed in liquid nitrogen and assayed for {beta}-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.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Localization to viroplasms
With the aim of investigating the localization of NSP5 and NSP2 to viroplasms, we constructed NSP2–EGFP (enhanced green fluorescent protein) and NSP5–EGFP fusion proteins (in both cases at the N terminus of EGFP) and used them to obtain stable transfectants, which were subsequently infected with rotavirus. As shown in Fig. 1(A), virus infection induced a rapid redistribution of the fusion proteins, with localization to viroplasms. However, while NSP2 appeared to occupy the central part of the viroplasms (Fig. 1B), NSP5 was always visualized in a more external part as shown by the ring structure formed. This result was obtained using either NSP2 or NSP5 fused to EGFP with the corresponding partner protein visualized with a specific antibody. Similar results were obtained in non-transfected virus-infected cells using antibodies specific for NSP2 and NSP5, which showed that the two proteins co-localized, although a ring of NSP5 was still visible. These results were obtained with both SA11 and OSU viral strains, suggesting that they were independent of NSP6, a 93 amino acid protein encoded as an alternative ORF in SA11 gs11 mRNA, which is truncated in OSU (Mattion et al., 1991).



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 1. (A) MA104 cells stably transfected with NSP2-EGFP or NSP5-EGFP were infected with rotavirus and analysed at 4 h post-infection. Bars, 10 µm. (B) Amplified images of viroplasms in rotavirus-infected cells. Upper panel, cells expressing NSP2–EGFP visualized with anti-NSP5 serum (red); middle panel, cells expressing NSP5-EGFP visualized with anti-NSP2 serum (red); lower panel, double immunofluorescence in non-transfected infected cells visualized with anti-NSP5 (red) and anti-NSP2 (green) sera. Images were obtained by confocal microscopy. Bars, 2 µm.

 
Kinetics of viroplasm formation
We took advantage of the NSP2–EGFP stably transfected cells to follow, by confocal microspcopy, the assembly of viroplasms at various time points post-infection (from 2 to 24 h). Infection was also assessed by immunofluorescence with a specific anti-NSP5 antibody (Fig. 2A, red). The number of viroplasms was counted and their area determined using a specific option in the confocal microscope programme available in the instrument. While the area of single viroplasms increased with time from 6 h post-infection, the total number of viroplasms per cell diminished (Fig. 2B). These results suggested a fusion between different viroplasms, supported by the fact that the shape of some of them appeared to be bean-like at 6 h post-infection (Fig. 2C). In addition, a three-dimensional reconstruction of stacked images obtained at different levels in the horizontal plane (Fig. 3) suggested that the viroplasms were spherical structures by 24 h post-infection.



View larger version (66K):
[in this window]
[in a new window]
 
Fig. 2. (A) Viroplasms visualized by confocal microscopy at various times post-infection in cells expressing NSP2–EGFP and infected with rotavirus. NSP5 was detected with anti-NSP5 serum (red). Bars, 10 µm. (B) Plot of the number and mean area of viroplasms, determined at various times post-infection. Each value corresponds to the mean of 20 cells counted. {circ}, Viroplasm area; {bullet}, number of viroplasms. (C) Bean-shaped viroplasms at 6 h post-infection (arrow). Bars, 2 µm.

 


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. Horizontal stack images of viroplasms in cells expressing NSP2–EGFP and infected with rotavirus, examined at 24 h post-infection. NSP5 is visualized in red. Numbers indicate the order of the stacks. Bars, 2 µm.

 
Yeast two-hybrid interaction between NSP5 mutants and NSP2
To characterize further the NSP2–NSP5 interaction, we used a yeast two-hybrid assay to identify the relevant NSP5 domains involved. The NSP5 deletion mutants presented in Fig. 4 were used as bait, with NSP2 fused to the herpes simplex virus transactivator protein, VP16 (Visintin et al., 1999). Positive interaction was determined by growth in a medium lacking histidine followed by a {beta}-galactosidase assay. All baits used in this assay were first tested to rule out possible transactivation activity in the absence of NSP2 and their expression checked by Western blotting (data not shown). As a result, neither wtNSP5 nor mutant {Delta}3 could be used in this assay since they showed transactivating activity per se.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4. (A) Scheme of the NSP5 mutants fused to LexA used in the yeast two-hybrid assay. (B) Results of the interaction between NSP5 mutants and NSP2. +++, Cells grown in plates lacking histidine and positive in the {beta}-galactosidase assay (strong blue colour developed in 15 min); -, lack of growth in plates lacking histidine and negative for {beta}-galactosidase (no blue colour developed in 12 h) when grown in histidine-supplemented plates.

 
The results obtained are shown in Fig. 4. Mutants in which regions 2 or 4 were deleted could interact with NSP2, while mutants in which regions 1, 3 or the C-terminal T were deleted could not. These results indicated that the N- and C-terminal regions, as well as the central part (region 3, aa 81–130), of NSP5 play a crucial role in the interaction with NSP2.

Binding assay in mammalian cells
As an alternative method for studying NSP5–NSP2 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.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5. (A) Immunoprecipitation with anti-NSP2 serum of cellular extracts from [35S]methionine-labelled MA104 cells infected with rotavirus OSU (lane 1) or SA11 (lane 2), or transfected with pT7v-NSP2 and infected with vaccinia virus VVT7 (lane 3), or infected with recombinant rVV/NSP2 and non-induced (lane 4) or induced (lane 5) with 1 mM IPTG. (B) In vivo binding/immunoprecipitation assay. Electrophoresis of extracts of cells infected with rVV/NSP2 at an m.o.i. of 3 and IPTG induced, transfected with the indicated NSP5 mutants, labelled with [35S]methionine and immunoprecipitated with anti-NSP5. (C) NSP2 binding activity of NSP5 mutants plotted relative to wtNSP5, which was defined as 1. The mean value for each mutant was obtained from three independent experiments.

 
To analyse the binding of NSP2 to NSP5, cells were infected with rVV/NSP2 at a multiplicity of 3 p.f.u. per cell, transfected with the different NSP5 deletion mutant constructs, induced with 1 mM IPTG and labelled overnight with [35S]methionine. To stabilize the interaction, chemical cross-linking with DSP was performed in living cells, followed by lysis and immunoprecipitation with anti-NSP5. The relative binding was determined by densitometry of the bands obtained, following autoradiography of the SDS-polyacrylamide gel. The ratio was calculated as described in the Methods.

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 {Delta}1, {Delta}1/{Delta}2 and {Delta}1/{Delta}3) had a profound effect on the ability to bind NSP2. Similarly, deletion of the C-terminal region T (mutants {Delta}T, {Delta}4T, {Delta}2/{Delta}4T and {Delta}3/{Delta}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 {Delta}3 showed a relative binding twofold higher than wtNSP5. However, when regions 1 or T from this mutant were also deleted ({Delta}1/{Delta}3 or {Delta}3/{Delta}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).



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 6. Viroplasm localization and VLS formation of EGFP fused to the N- and C-terminal regions of NSP5. Viroplasm localization was determined at 4 h post-infection (left), while formation of VLSs was determined at 18 h post-transfection of cells infected with rVV/NSP2 and IPTG induced, and immediately transfected with the indicated constructs (right). Bars, 20 µm.

 
Table 1 summarizes the interactions of EGFP–NSP5 domain fusion constructs in terms of localization to the viroplasm in virus-infected cells and formation of VLSs when co-transfected with NSP2. These results are consistent with those obtained with the yeast two-hybrid analysis and the in vivo binding/immunoprecipitation assay, in which the N-terminal region and the C-terminal tail of NSP5 were characterized as the two regions necessary for NSP2 binding.


View this table:
[in this window]
[in a new window]
 
Table 1. Interactions of the EGFP–NSP5 domain fusion constructs

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The non-fusogenic mammalian orthoreoviruses replicate and assemble in cytoplasmic phase-dense inclusions in infected cells. These inclusions contain viral dsRNA (Silverstein & Schur, 1970), viral proteins and partially assembled viral particles (Rhim et al., 1962; Dales et al., 1965). In the case of rotaviruses, the inclusion bodies, called viroplasms, are globular structures identified as the machinery of virus replication. Immunogold staining with antibodies against NSP2 and NSP5 has suggested that both proteins localize in the external part of the globular structure of the viroplasms (Petrie et al., 1984). Here, we used confocal microscopy to investigate the localization in viroplasms of NSP2 and NSP5 using stable transfectants of NSP5–EGFP and NSP2–EGFP, followed by rotavirus infection. Magnification of the viroplasm images showed NSP2 and NSP5 co-localization, although NSP5 appeared relatively concentrated in a more external region with respect to NSP2. There are at least two possible interpretations of this observation: (i) it represents a relative different distribution of the two proteins within viroplasms; or (ii) it is the consequence of different reactivity or accessibility of the proteins to the antibodies. In fact, in other experiments, co-immunoprecipitation carried out with anti-NSP2 or anti-NSP5 showed that, while anti-NSP5 was able to co-precipitate NSP2, anti-NSP2 was not (Afrikanova et al., 1998; C. Eichwald & O. R. Burrone, unpublished data). In addition, in double immunofluorescence studies we have observed that incubating first with an anti-NSP5 serum blocks binding of anti-NSP2, while the reverse does not (data not shown). Further studies are needed to clarify this issue.

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 {sigma}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 ({Delta}3) was not possible because the construct LexA–{Delta}3 was transactivating in the absence of NSP2–VP16. This was also the case for wtNSP5 and mutant {Delta}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).


   ACKNOWLEDGEMENTS
 
C. E. was supported by a SISSA pre-doctoral fellowship. We are grateful to Fulvia Vascotto for the support with the yeast two-hybrid system.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Afrikanova, I., Miozzo, M. C., Giambiagi, S. & Burrone, O. (1996). Phosphorylation generates different forms of rotavirus NSP5. J Gen Virol 77, 2059–2065.[Abstract]

Afrikanova, I., Fabbretti, E., Miozzo, M. C. & Burrone, O. R. (1998). Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2. J Gen Virol 79, 2679–2686.[Abstract]

Becker, M. M., Peters, T. R. & Dermody, T. S. (2003). Reovirus {sigma}NS and µNS proteins form cytoplasmic inclusion structures in the absence of viral infection. J Virol 77, 5948–5963.[Abstract/Free Full Text]

Berois, M., Sapin, C., Erk, I., Poncet, D. & Cohen, J. (2003). Rotavirus nonstructural protein NSP5 interacts with major core protein VP2. J Virol 77, 1757–1763.[Abstract/Free Full Text]

Blackhall, J., Munoz, M., Fuentes, A. & Magnusson, G. (1998). Analysis of rotavirus nonstructural protein NSP5 phosphorylation. J Virol 72, 6398–6405.[Abstract/Free Full Text]

Breeden, L. & Nasmyth, K. (1985). Regulation of the yeast HO gene. Cold Spring Harbor Symp Quant Biol 50, 643–650.[Medline]

Dales, S., Gomatos, P. & Hsu, K. (1965). The uptake and development of reovirus in strain L cells followed with labelled viral riboznucleic acid and ferritin-antibody complexes. Virology 25, 193–211.[CrossRef][Medline]

Earl, P. L. & Moss, B. (1993). Generation of recombinant vaccinia viruses. In Current Protocols in Molecular Biology. Edited by F. M. B. Ausubel, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Wiley & Sons.

Eichwald, C., Vascotto, F., Fabbretti, E. & Burrone, O. R. (2002). Rotavirus NSP5: mapping phosphorylation sites and kinase activation and viroplasm localization domains. J Virol 76, 3461–3470.[Abstract/Free Full Text]

Estes, M. K., Graham, D. Y., Gerba, C. P. & Smith, E. M. (1979). Simian rotavirus SA11 replication in cell cultures. J Virol 31, 810–815.[Medline]

Fabbretti, E., Afrikanova, I., Vascotto, F. & Burrone, O. R. (1999). Two non-structural rotavirus proteins, NSP2 and NSP5, form viroplasm-like structures in vivo. J Gen Virol 80, 333–339.[Abstract]

Fillmore, G. C., Lin, H. & Li, J. K. (2002). Localization of the single-stranded RNA-binding domains of bluetongue virus nonstructural protein NS2. J Virol 76, 499–506.[Abstract/Free Full Text]

Gallegos, C. O. & Patton, J. T. (1989). Characterization of rotavirus replication intermediates: a model for the assembly of single-shelled particles. Virology 172, 616–627.[Medline]

Gietz, D., St Jean, A., Woods, R. A. & Schiestl, R. H. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20, 1425.[Medline]

Gillian, A. L. & Nibert, M. L. (1998). Amino terminus of reovirus nonstructural protein sigma NS is important for ssRNA binding and nucleoprotein complex formation. Virology 240, 1–11.[CrossRef][Medline]

Gillian, A. L., Schmechel, S. C., Livny, J., Schiff, L. A. & Nibert, M. L. (2000). Reovirus protein {sigma}NS binds in multiple copies to single-stranded RNA and shares properties with single-stranded DNA binding proteins. J Virol 74, 5939–5948.[Abstract/Free Full Text]

Gonzalez, S. A. & Burrone, O. R. (1991). Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine. Virology 182, 8–16.[Medline]

Grimley, P. M., Rosenblum, E. N., Mims, S. J. & Moss, B. (1970). Interruption by rifampin of an early stage in vaccinia virus morphogenesis: accumulation of membranes which are precursors of virus envelopes. J Virol 6, 519–533.[Medline]

Huismans, H. & Joklik, W. K. (1976). Reovirus-coded polypeptides in infected cells: isolation of two native monomeric polypeptides with affinity for single-stranded and double-stranded RNA, respectively. Virology 70, 411–424.[Medline]

Jayaram, H., Taraporewala, Z., Patton, J. T. & Prasad, B. V. (2002). Rotavirus protein involved in genome replication and packaging exhibits a HIT-like fold. Nature 417, 311–315.[CrossRef][Medline]

Kattoura, M. D., Clapp, L. L. & Patton, J. T. (1992). The rotavirus nonstructural protein, NS35, possesses RNA-binding activity in vitro and in vivo. Virology 191, 698–708.[Medline]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]

Mattion, N. M., Mitchell, D. B., Both, G. W. & Estes, M. K. (1991). Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11. Virology 181, 295–304.[Medline]

Nagaya, A., Pogo, B. G. & Dales, S. (1970). Biogenesis of vaccinia: separation of early stages from maturation by means of rifampicin. Virology 40, 1039–1051.[Medline]

Patton, J. T. & Gallegos, C. O. (1990). Rotavirus RNA replication: single-stranded RNA extends from the replicase particle. J Gen Virol 71, 1087–1094.[Abstract]

Patton, J. T., Jones, M. T., Kalbach, A. N., He, Y. W. & Xiaobo, J. (1997). Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome. J Virol 71, 9618–9626.[Abstract]

Petrie, B. L., Greenberg, H. B., Graham, D. Y. & Estes, M. K. (1984). Ultrastructural localization of rotavirus antigens using colloidal gold. Virus Res 1, 133–152.[CrossRef][Medline]

Poncet, D., Lindenbaum, P., L’Haridon, R. & Cohen, J. (1997). In vivo and in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms. J Virol 71, 34–41.[Abstract]

Rhim, J., Jordan, L. & Mayor, H. (1962). Cytochemical, fluorescent-antibody and electron microscopic studies on the growth of reovirus (ECHO 10) in tissue culture. Virology 17, 342–355.[Medline]

Richardson, M. A. & Furuichi, Y. (1985). Synthesis in Escherichia coli of the reovirus nonstructural protein sigma NS. J Virol 56, 527–533.[Medline]

Sambrook, J., Fristisch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor.

Schuck, P., Taraporewala, Z., McPhie, P. & Patton, J. T. (2001). Rotavirus nonstructural protein NSP2 self-assembles into octamers that undergo ligand-induced conformational changes. J Biol Chem 276, 9679–9687.[Abstract/Free Full Text]

Silverstein, S. C. & Schur, P. H. (1970). Immunofluorescent localization of double-stranded RNA in reovirus-infected cells. Virology 41, 564–566.[Medline]

Taraporewala, Z. F. & Patton, J. T. (2001). Identification and characterization of the helix-destabilizing activity of rotavirus nonstructural protein NSP2. J Virol 75, 4519–4527.[Abstract/Free Full Text]

Taraporewala, Z. F., Chen, D. & Patton, J. T. (2001). Multimers of the bluetongue virus nonstructural protein, NS2, possess nucleotidyl phosphatase activity: similarities between NS2 and rotavirus NSP2. Virology 280, 221–231.[CrossRef][Medline]

Taraporewala, Z. F., Schuck, P., Ramig, R. F., Silvestri, L. & Patton, J. T. (2002). Analysis of a temperature-sensitive mutant rotavirus indicates that NSP2 octamers are the functional form of the protein. J Virol 76, 7082–7093.[Abstract/Free Full Text]

Torres-Vega, M. A., Gonzalez, R. A., Duarte, M., Poncet, D., Lopez, S. & Arias, C. F. (2000). The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6. J Gen Virol 81, 821–830.[Abstract/Free Full Text]

Vende, P., Taraporewala, Z. F. & Patton, J. T. (2002). RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA. J Virol 76, 5291–5299.[Abstract/Free Full Text]

Visintin, M., Tse, E., Axelson, H., Rabbitts, T. H. & Cattaneo, A. (1999). Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc Natl Acad Sci U S A 96, 11723–11728.[Abstract/Free Full Text]

Ward, G. A., Stover, C. K., Moss, B. & Fuerst, T. R. (1995). Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells. Proc Natl Acad Sci U S A 92, 6773–6777.[Abstract]

Welch, S. K., Crawford, S. E. & Estes, M. K. (1989). Rotavirus SA11 genome segment 11 protein is a nonstructural phosphoprotein. J Virol 63, 3974–3982.[Medline]

Wentz, M. J., Patton, J. T. & Ramig, R. F. (1996). The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis. J Virol 70, 7833–7841.[Abstract]

Zeng, C. Q., Estes, M. K., Charpilienne, A. & Cohen, J. (1998). The N terminus of rotavirus VP2 is necessary for encapsidation of VP1 and VP3. J Virol 72, 201–208.[Abstract/Free Full Text]

Received 29 August 2003; accepted 13 November 2003.