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Reovirus NS Protein Localizes to Inclusions through an Association Requiring the µNS Amino Terminus

Department of Microbiology and Molecular Genetics,¹ Virology Training Program, Harvard Medical School, Boston, Massachusetts 02115²

Received 11 October 2002/ Accepted 23 January 2003

	ABSTRACT

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Cells infected with mammalian reoviruses contain phase-dense inclusions, called viral factories, in which viral replication and assembly are thought to occur. The major reovirus nonstructural protein µNS forms morphologically similar phase-dense inclusions when expressed in the absence of other viral proteins, suggesting it is a primary determinant of factory formation. In this study we examined the localization of the other major reovirus nonstructural protein, NS. Although NS colocalized with µNS in viral factories during infection, it was distributed diffusely throughout the cell when expressed in the absence of µNS. When coexpressed with µNS, NS was redistributed and colocalized with µNS inclusions, indicating that the two proteins associate in the absence of other viral proteins and suggesting that this association may mediate the localization of NS to viral factories in infected cells. We have previously shown that µNS residues 1 to 40 or 41 are both necessary and sufficient for µNS association with the viral microtubule-associated protein µ2. In the present study we found that this same region of µNS is required for its association with NS. We further dissected this region, identifying residues 1 to 13 of µNS as necessary for association with NS, but not with µ2. Deletion of NS residues 1 to 11, which we have previously shown to be required for RNA binding by that protein, resulted in diminished association of NS with µNS. Furthermore, when treated with RNase, a large portion of NS was released from µNS coimmunoprecipitates, suggesting that RNA contributes to their association. The results of this study provide further evidence that µNS plays a key role in forming the reovirus factories and recruiting other components to them.

	INTRODUCTION

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The nonfusogenic mammalian orthoreoviruses (reoviruses) are double-stranded (ds) RNA viruses that contain 10 genome segments encased by a multilayered protein capsid (reviewed in reference 26). Following cell entry, plus-sense RNAs representing full-length copies of each of the 10 segments are transcribed and capped by virally encoded enzymes within the infecting particle (primary transcriptase particle) (7, 13, 15, 33). Following extrusion into the cytoplasm (3, 4), these primary transcripts serve both as mRNAs for viral protein translation and as templates for minus-strand synthesis to regenerate the dsRNA genome segments within newly forming particles (1, 31, 34, 41). At least some of these new particles can act as secondary transcriptase particles, producing additional large amounts of the plus-strand transcripts (27, 36, 40). The interactions between viral proteins and RNAs that recruit the necessary components and form the sites of minus-strand synthesis and core assembly have only begun to be elucidated. Furthermore, the involvement of host proteins in these processes has not been well explored.

The replication and assembly of reoviruses occur within distinct structures called viral factories that appear as small phase-dense inclusions throughout the cytoplasm early in infection and become larger and move toward the nucleus as infection proceeds (5, 28, 29). Viral factories are not membrane bound (25) but associate with cytoskeletal elements such as microtubules (11, 12, 28). The viral dsRNA (35), many of the proteins, and partially and fully assembled particles have been localized to the factories (12, 29). However, the assembly and inner workings of the factories are still poorly understood. A viral strain difference in the formation of filamentous versus globular factories has been recently mapped to the reovirus core protein µ2 (28). µ2 from strains that form filamentous factories associates with and stabilizes microtubules, and the association between µ2 and microtubules has been proposed to determine the morphology of the filamentous viral factories by anchoring them to the microtubule network in infected cells (28). A viral strain difference in the rate of inclusion formation has also been mapped to the µ2 protein (25a).

µNS, a major reovirus nonstructural protein, has been recently implicated in viral factory formation (9). µNS is a 721-residue, 80-kDa protein encoded by the M3 genome segment. A second form of µNS, µNSC, thought to lack 40 residues from the amino (N) terminus of µNS, is also detected during infection (38). µNS binds to core particles in vitro, forming large complexes that remain competent for transcription and capping of the viral plus-strand RNAs (8). The bound µNS prevents outer-capsid proteins from recoating the cores, which suggests that during infection µNS may be involved in delaying outer-capsid assembly so that larger amounts of the transcripts can be produced by these particles (8). When expressed in the absence of other viral proteins, µNS forms globular inclusions that are morphologically similar to the globular viral factories seen during infection with certain reovirus strains (9). In addition, µ2 proteins derived from viral strains with either filamentous or globular factories associate with the N-terminal 40 or 41 residues of µNS. When coexpressed with the filamentous form of µ2, µNS colocalizes with µ2 on microtubules in a pattern very similar to that seen during infection (9). These findings have led us to hypothesize that while µ2 plays an important role in anchoring the factories to microtubules, µNS is the primary determinant of factory formation and may be involved in recruiting other components required for RNA assortment, minus-strand synthesis, and core assembly (9).

A second major reovirus nonstructural protein, NS, has also been implicated in viral factory formation (5, 25a). NS is a 366-residue, 41-kDa protein encoded by the S3 genome segment. In vitro, NS binds single-stranded (ss) RNA in a cooperative, sequence-independent manner, with each unit of NS covering 25 nucleotides (17, 37). When isolated from reovirus-infected cells, it is found in large (40- to 60S) complexes that dissociate when treated with RNase A (18, 19, 22), suggesting that NS and RNA form large nucleoprotein complexes during infection. Recombinant NS expressed in baculovirus-infected cells is found in similar large nucleoprotein complexes, which dissociate into 7- to 9S complexes upon treatment with RNase A or high-salt washes, suggesting that NS forms small oligomers in the absence of RNA (16). NS and µNS, as well as the structural protein 3, have been isolated from infected cells in complexes with viral ssRNA, leading to the proposal that these proteins are involved in preparing the viral transcripts for minus-strand synthesis and packaging into progeny cores (2). NS has been recently proposed to nucleate viral factories, based on evidence that it localizes to factories throughout infection and that a viral mutant with a temperature-sensitive NS protein (tsE320) is defective for factory formation at restrictive temperatures (5).

To address our hypothesis that µNS may be involved in recruiting other components to reovirus factories, we examined NS localization when expressed with µNS in the absence of other viral proteins. We found a strong association between NS and µNS and identified that the N-terminal 40 residues of µNS, which our laboratory has previously shown to be necessary for association with µ2 (9), are also necessary for the association with NS. This supports our hypothesis that µNSC, which is thought to lack this N-terminal region, plays some role(s) distinct from those of µNS during infection. We further dissected this region, identifying residues 1 to 13 of µNS as necessary for association with NS, but not with µ2. Deletion of NS residues 1 to 11, which our laboratory has previously shown to be required for RNA binding by that protein (16), resulted in diminished association of NS with µNS. Furthermore, when treated with RNase, a large portion of NS was released from µNS coimmunoprecipitates, suggesting that RNA contributes to their association. The results of this study provide further strong evidence that µNS plays a key role in forming the reovirus factories and recruiting other components to them.

	MATERIALS AND METHODS

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Cells and viruses. CV-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Life Technologies, Carlsbad, Calif.) containing 10% fetal bovine serum (HyClone Laboratories, Logan, Utah) and 10 µg of gentamicin (Invitrogen)/ml. Reovirus strains T1L and T3D^N were our laboratory stocks. The designation T3D^N differentiates our laboratory strain from another T3D laboratory strain (T3D^C) that has a filamentous viral factory phenotype.

Antibodies and other reagents. Mouse monoclonal antibody (MAb) 3E10 specific for NS (5) was a generous gift from T. S. Dermody and colleagues (Vanderbilt University, Nashville, Tenn.). Rabbit polyclonal antisera specific for µNS (8), µ2 (9), and NS (16) have been described previously. In some experiments, Texas Red conjugates of polyclonal antibodies purified from the µNS antiserum were used (preparation described previously [28]). The following secondary antibodies were used as appropriate for different experiments: Alexa 488- or Alexa 594-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (IgG) (Molecular Probes, Eugene, Oreg.), horseradish peroxidase (HRP)-conjugated donkey anti-mouse or anti-rabbit IgG (Pierce, Rockford, Ill.), and alkaline phosphatase-coupled goat anti-mouse or anti-rabbit IgG (Bio-Rad Laboratories, Hercules, Calif.). For microscopy, antibodies were titrated to optimize signal-to-noise ratios. All restriction enzymes were obtained from New England Biolabs (Beverly, Mass.).

Plasmid constructs. All reovirus genes examined in this study were cloned into pCI-neo (Promega, Madison, Wis.). pCI-M1(T1L) (28), pCI-M1(T3D^N) (28), pCI-M3(T1L) (9), and pCI-M3(41-721) (9) were previously described. The T1L S3 gene was excised from pGEM-4Z:LS3 (16) with HindIII and EcoRI. The HindIII end was converted to a blunt end using the Klenow fragment. The T1L S3 gene was then ligated to pCI-neo that had been cut with NheI and EcoRI and had its NheI end converted to a blunt end. This procedure generated pCI-S3(T1L). The T3D S3 gene was amplified by reverse transcription-PCR from reovirus transcripts made from T3D cores (20) by using a forward primer with an XbaI site (5'-GGTCTAGATGATTAGGCGTCACCC-3') and a reverse primer containing an EcoRI site (5'-GGGAATTCGCTAAAGTCACGCCTTGTCGTCG-3'). The PCR product and pCI-neo were each digested with XbaI and EcoRI and then ligated to generate pCI-S3(T3D). To obtain the S3 gene of temperature-sensitive mutant tsE320 (14), overlap-PCR mutagenesis was performed (21). A forward mutagenic primer (5'-GTGTTAAATTGCACGCAGTTTAAACTTGAG-3') and reverse mutagenic primer (5'-CTCAAGTTTAAACTGCGTGCAATTTAACAC-3') and the above forward and reverse primers were used to PCR amplify an S3 gene fragment encoding a threonine at amino acid 260 (39). The purified fragment and pCI-S3(T3D) were digested with XbaI and EcoRI, gel purified, and ligated to generate pCI-S3(M260T). To express the T1L NS protein lacking amino acids 1 to 11, the mutated T1L S3 gene was removed from the previously constructed pGEM-4Z.LS3.XhoI.31-60 (16) with XhoI and KpnI. The mutated T1L S3 gene was ligated to pCI-neo cut with XhoI and KpnI. This procedure generated pCI-S3(12-366). The portion of the T1L M3 genome segment encoding µNS amino acids 14 to 721 was amplified by PCR with a forward primer containing an NheI site and a methionine codon (5'-GACTGCTAGCATGGTTTCCAAGGCCAAACGTGATATATCATCTCTGCC-3') and a reverse primer (5'-GGCATATAGGTCATCAGGCACAGAGCG-3') containing a BlpI site. The purified product was cut with NheI and BlpI and ligated to pCI-M3(T1L) that had been cut with NheI and BlpI to generate pCI-M3(14-721). The ATG codon introduced at nucleotides 55 to 57 (amino acid 13) in the M3 gene allowed expression of µNS amino acids 14 to 721. The sequences of all portions of plasmids amplified by PCR were determined to ensure they matched those published.

Transfections and infections. For immunostaining experiments, 1.5 x 10⁵ CV-1 cells were seeded the day before infection or transfection in six-well plates (9.6 cm² per well) containing 18-mm round glass coverslips. A total of 2 µg of DNA was transfected into cells using 7 µl of Lipofectamine (Invitrogen) in Optimem (Invitrogen) and incubated for 4 h as suggested by the manufacturer. After incubation, fresh DMEM was added to the cells, and they were incubated at 37°C for a further 14 h before processing for immunofluorescence (IF) microscopy. For immunoprecipitation (IP) studies, 3 x 10⁵ cells were seeded onto 60-mm dishes the day before transfection or infection. Two micrograms of DNA was transfected into cells by using 9 µl of Lipofectamine in Optimem and incubating for 4 h at 37°C. After incubation, fresh DMEM was added to the cells, and they were incubated for a further 14 h at 37°C before preparing for IP. For both immunostaining and IP experiments, cells in dishes or on coverslips were inoculated with T1L or T3D^N reovirus at a multiplicity of 10 PFU per cell in phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄ [pH 7.5]) supplemented with 2 mM MgCl₂. The virus was adsorbed for 1 h at room temperature, at which point fresh medium was added. Infected cells were then incubated at 37°C for an additional 5 to 19 h before processing for IF microscopy or IP.

Immunostaining and IF microscopy. Infected or transfected cells were fixed by incubation at room temperature with 2% paraformaldehyde in PBS for 10 min, followed by 3 min at -20°C in 100% ice-cold methanol. Fixed cells were washed three times in PBS and permeabilized for 5 min in 0.2% Triton X-100 in PBS. Cells were again washed three times in PBS and blocked for 5 min in 0.1 M glycine in PBS. Primary and secondary antibodies were diluted in 0.1 M glycine in PBS. After blocking, cells were incubated for 1 h with primary antibodies, washed three times in PBS, and then incubated with secondary antibodies for 1 h. Immunostained cells were washed a final three times with PBS, incubated for 5 min in 300 nM 4',6-diamidino-2-phenylindole, and mounted on slides with Prolong reagent (Molecular Probes). Immunostaining was alternatively performed using 1% bovine serum albumin in PBS as a blocking agent. Immunostained samples were examined using a Nikon TE-300 inverted microscope with fluorescence optics. Images were collected digitally as described elsewhere (28) and prepared for presentation using Photoshop and Illustrator software (Adobe Systems, San Jose, Calif.).

IP analysis. Infected or transfected cells were lysed by incubation for 30 min on ice in nondenaturing lysis (Raf) buffer (20 mM Tris [pH 8.0], 137 mM NaCl, 10% glycerol, 1% NP-40) (6) containing protease inhibitors (Roche Biochemicals, Indianapolis, Ind.). Because of nonspecific binding to protein A-conjugated beads by many of the proteins in this study, lysates were precleared for 1 h by incubation with 50 µl of a 50:50 slurry of protein A-Sepharose beads in Raf buffer, or with 50 µl of a 50:50 slurry of protein A-Sepharose beads in Raf buffer that were prebound to µNS preimmune antibody (9). After centrifugation at 13,000 x g to pellet protein A-conjugated beads and cell debris, lysates were transferred to new tubes. The protein concentration of each lysate was measured by Bradford assay (Bio-Rad) and normalized for relative protein concentration within each experiment. Immunoprecipitating antibodies that had been incubated for 2 h with protein A-conjugated magnetic beads (Dynal Biotech, Lake Success, N.Y.) and washed six times with Raf buffer were then added to the cell lysates, which were incubated, rotating, overnight at 4°C. Immunoprecipitated proteins were washed four times with Raf buffer and resuspended in sample buffer (125 mM Tris [pH 6.8], 10% sucrose, 1% [wt/vol] sodium dodecyl sulfate [SDS], 0.02% [vol/vol] ß-mercaptoethanol, 0.01% bromophenol blue).

Immunoblot analysis. Immunoprecipitated proteins prepared as described above were boiled for 3 min and separated on SDS-10% polyacrylamide gel electrophoresis (PAGE) gels. Proteins were transferred to nitrocellulose by electroblotting in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol [pH 8.3]). Binding of primary antibodies was detected with HRP-conjugated secondary antibodies and Supersignal chemiluminescence reagent (Pierce). Supersignal-treated immunoblots were exposed to film to visualize bound HRP conjugates. Alternatively, binding of primary antibodies was detected with alkaline phosphatase-conjugated secondary antibodies and the colorimetric reagents p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Bio-Rad).

RNase ONE treatment. Proteins (with associated antibodies and protein A beads) immunoprecipitated as described above were split into two equal aliquots and resuspended in 10 µl of 10 mM Tris (pH 7.5). RNase ONE buffer (10 mM Tris-HCl [pH 7.5], 5 mM EDTA, 200 mM sodium acetate) was added to a 1x final concentration to both aliquots. Ten units of RNase ONE (Promega) was added to one aliquot, and both samples were incubated at 37°C for 30 min (23). After centrifugation at 13,000 x g, supernatants were removed from the pellets and sample buffer was added to both supernatants and pellets, which were then boiled and loaded on SDS-PAGE gels.

	RESULTS

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NS and µNS colocalize in viral factories during reovirus infection. Our laboratory has recently shown that nonstructural protein µNS localizes to, and is likely involved in the formation of, viral factories during reovirus infection (9). Other previous studies have indicated that the nonstructural protein NS also localizes to these factories (5). To determine whether NS and µNS colocalize in the factories, we examined the subcellular localization of NS and µNS in T1L- and T3D^N-infected CV-1 cells by immunostaining with protein-specific antibodies at different times postinfection (p.i.). From the earliest time points that NS and µNS were readily detectable (6 to 8 h p.i.), both proteins were found in a similar punctate pattern, with obvious regions of colocalization, throughout the cytoplasm (Fig. 1A, first and third rows). As infection proceeded, NS and µNS continued to colocalize in the factories as they grew in size and moved to a perinuclear location (Fig. 1A, second and fourth rows). A difference in T1L (filamentous) and T3D^N (globular) factory morphologies at 18 to 24 h p.i., which our laboratory has recently mapped to the M1 genome segment (28), was apparent in these experiments; however, this difference had no detectable effect on NS and µNS colocalization, in that the two proteins colocalized in both types of factories (Fig. 1A, second and fourth rows). These findings suggest that a large portion of the NS and µNS proteins in cells colocalize throughout infection.

View larger version (48K): [in this window] [in a new window]   FIG. 1. Colocalization and co-IP of NS and µNS in T1L- and T3DN-infected CV-1 cells. (A) IF microscopy of CV-1 cells infected with reovirus T1L (top two rows) or T3DN (bottom two rows) at 6 h p.i. (first and third rows) or 20 h p.i. (second and fourth rows). The subcellular localizations of NS and µNS were, respectively, detected by immunostaining with NS-specific mouse MAb 3E10 (5) followed by Alexa 488-conjugated anti-mouse IgG (left column) and Texas Red-conjugated µNS-specific polyclonal antibodies (9) (right column). Insets show higher-magnification views of the NS and µNS staining patterns at 6 h p.i. Scale bars, 10 µm. (B) Co-IP of NS and µNS from T1L- and T3DN-infected CV-1 cells. At 18 h p.i., T1L-, T3DN-, or mock-infected CV-1 cells were lysed in nondenaturing buffer and immunoprecipitated (IP) with µNS-specific rabbit polyclonal antiserum (8) (left) or NS MAb (right). Protein A-Sepharose beads alone (BA) or with rabbit preimmune serum (PI) were used as controls. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with NS-specific rabbit polyclonal antiserum (16) followed by HRP-conjugated anti-rabbit IgG (left) or with µNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence. The background levels of NS and µNS in the BA and PI lanes represent nonspecific binding of these proteins to the beads.

NS and µNS are coimmunoprecipitated from infected cells.

NS does not form inclusions when expressed alone in transfected cells. Previous reports have suggested that NS nucleates viral factories (5). To determine if NS forms factory-like inclusions when expressed in the absence of other viral proteins, we transfected CV-1 cells with pCI-S3(T1L) or pCI-S3(T3D). At 18 h posttransfection (p.t.), NS was visualized by immunostaining with NS MAb. NS expressed from either the T1L S3 gene (Fig. 2A, top left) or the T3D S3 gene (http://micro.med.harvard.edu/nibert/suppl/miller03a/Fig.1.html) exhibited a diffuse distribution throughout the cytoplasm, with occasional cells also showing punctate staining in the nucleus. While these results do not rule out the possibility that NS participates in forming the viral factories during infection, the diffuse cytoplasmic pattern of NS staining seen here is inconsistent with NS nucleating the factories independently of other viral components.

View larger version (60K): [in this window] [in a new window]   FIG. 2. Colocalization and co-IP of NS and µNS in transfected CV-1 cells. (A) IF microscopy of CV-1 cells at 18 h p.t. with pCI-S3 (top left), pCI-M3 (top right), or both pCI-S3 and pCI-M3 (bottom row). NS was visualized by immunostaining with NS-specific mouse MAb 3E10 (5) followed by Alexa 488-conjugated anti-mouse IgG (left column). µNS was detected by immunostaining with Texas Red-conjugated µNS-specific polyclonal antibodies (9) (right column). Scale bars, 10 µm. (B) CV-1 cells transfected with pCI-neo (Vector), pCI-S3, pCI-M3, or both pCI-S3 and pCI-M3 were lysed in nondenaturing buffer and immunoprecipitated (IP) using NS MAb. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using µNS-specific rabbit polyclonal antiserum (8) followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.

NS is recruited to µNS inclusions when coexpressed in transfected cells.

NS and µ2 association in transfected cells. Our laboratory has recently identified the µ2 protein encoded by the reovirus M1 genome segment as a strain-specific microtubule-binding protein (28; J. Kim, J. S. L. Parker, and M. L. Nibert, submitted for publication). A difference in morphology of viral factories has also been mapped to the M1 segment, with M1(T1L)-containing viruses forming filamentous factories and M1(T3D^N)-containing viruses forming globular factories (28). During T1L infection, NS localizes to the filamentous factories (Fig. 1A, second row), which suggests that NS might associate with µ2 as well as µNS. To determine if NS colocalizes with µ2 in cells, we cotransfected CV-1 cells with pCI-S3 and pCI-M1(T1L) and examined the localization of both encoded proteins by immunostaining with protein-specific antibodies. When NS was coexpressed with µ2(T1L), the µ2 protein was localized to thin filaments previously shown to be microtubules (28) (Fig. 3A, top right), while NS retained a mostly diffuse staining pattern throughout the cytoplasm (Fig. 3A, top left). Although these results suggest that NS does not colocalize with µ2, we sometimes observed faint filamentous staining of NS in these experiments (Fig. 3A, top left) and were therefore prompted to examine further for an association with µ2 by determining whether NS and µ2 could be coimmunoprecipitated. CV-1 cells were infected with T1L, and at 18 h p.i. nondenaturing IP using either µ2 antiserum or NS MAb was performed on the cell lysates. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using protein-specific antibodies. µ2 was precipitated by µ2 antiserum, but not by NS MAb (Fig. 3B, left). Reciprocally, NS was precipitated by NS MAb, but not by µ2 antiserum (Fig. 3B, middle). As a control in this experiment, µNS was found to be coimmunoprecipitated by NS MAb (Fig. 3B, right), as already noted above (Fig. 1B). These results indicate that NS and µ2(T1L) do not strongly associate. Similar results were obtained using lysates from transfected CV-1 cells expressing the NS and µ2(T1L) proteins (data not shown). We also performed immunostaining to examine the localization of NS when coexpressed with µ2(T3D) and did not detect any colocalization between these proteins (http://micro.med.harvard.edu/nibert/suppl/miller03a/Fig. 2.html). We conclude that NS and µ2(T1L), when coexpressed in the absence of other viral proteins, might be weakly associated in cells but do not remain associated in cell lysates. Because µ2(T1L), but not µ2(T3D), induces microtubule bundling when expressed in cells (28), the faint filamentous pattern we observed when NS was coexpressed with µ2(T1L) might reflect NS association with bundled microtubules.

View larger version (46K): [in this window] [in a new window]   FIG. 3. µNS recruits NS to filamentous inclusion-like structures when coexpressed with T1L µ2. (A) IF microscopy of CV-1 cells transfected with both pCI-S3 and pCI-M1(T1L) (top row), both pCI-M3 and pCI-M1(T1L) (middle row), or pCI-S3, pCI-M3, and pCI-M1(T1L) (bottom row). NS was immunostained with NS-specific mouse MAb 3E10 (5) followed by Alexa 488-conjugated anti-mouse IgG (top left and bottom left). µNS was immunostained with Texas Red-conjugated µNS-specific polyclonal antibodies (9) (middle right and bottom right). µ2 was immunostained with µ2-specific rabbit polyclonal antiserum (9) followed by Alexa 594-conjugated anti-rabbit IgG (middle left and top right). Scale bars, 10 µm. (B) µ2 and NS do not coimmunoprecipitate. At 18 h p.i., T1L-infected CV-1 cells were lysed in nondenaturing buffer and immunoprecipitated (IP) using µ2 antiserum or NS MAb. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using µ2-specific (9) (left), NS-specific (16) (middle), or µNS-specific (8) (right) rabbit polyclonal antiserum followed by alkaline phosphatase-conjugated anti-rabbit IgG. Bound alkaline phosphatase conjugates were detected by colorimetric staining.

NS is localized to filamentous inclusions when coexpressed with µNS and T1L µ2.

µNS residues 1 to 40 are necessary for NS localization to µNS inclusions. During reovirus infection, a second protein related to µNS, µNSC, is detected (24). µNS and µNSC share the same open reading frame, but µNSC is thought to lack the 40 N-terminal residues from µNS (38). It is not known if µNSC expression is required or plays some role(s) independent of µNS during infection. Our recent studies have shown that µNS(41-721), a recombinant form of µNS lacking residues 1 to 40, forms globular inclusions similar to those of µNS when expressed in the absence of other viral proteins (9). However, whereas µNS colocalizes with µ2 following coexpression, µNS(41-721) does not (9). These results prompted us to examine the localization of NS in the presence of µNS(41-721) after cotransfecting CV-1 cells with pCI-S3 and pCI-M3(41-721) and immunostaining with protein-specific antibodies at 18 h p.t. As previously reported, the µNS(41-721) protein formed smooth-edged globular inclusions similar to those seen when µNS is expressed (Fig. 4A, right). The coexpressed NS protein, in contrast, was found diffusely distributed throughout the cytoplasm and did not colocalize with the µNS(41-721) inclusions (Fig. 4A), suggesting that the N-terminal 40 residues of µNS are required for recruiting NS. To examine further the lack of association between NS and µNS(41-721), we cotransfected pCI-S3 and pCI-M3(41-721) into CV-1 cells and performed IP with either µNS antiserum or NS MAb at 18 h p.t. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with µNS antiserum. Consistent with the immunostaining results, µNS, but no detectable µNS(41-721), coimmunoprecipitated with NS (Fig. 4B, right), even though similar amounts of µNS and µNS(41-721) were expressed (Fig. 4B, left). Several faster-mobility bands in both the µNS and the µNS(41-721) lanes likely resulted from protein degradation and did not coimmunoprecipitate with NS (Fig. 4B). These results indicate that the N-terminal 40 residues of µNS, previously shown to associate with µ2 (9), are also necessary for association with NS.

View larger version (67K): [in this window] [in a new window]   FIG. 4. µNS residues 1 to 40 are necessary for association with NS. (A) IF microscopy of CV-1 cells transfected with both pCI-S3 and pCI-M3(41-721). At 18 h p.t., NS and µNS(41-721) were, respectively, immunostained with NS-specific mouse MAb 3E10 (5) followed by Alexa 488-conjugated anti-mouse IgG (left column) and Texas Red-conjugated µNS-specific polyclonal antibodies (9) (right column). Scale bar, 10 µm. (B) µNS(41-721) does not coimmunoprecipitate with NS. At 18 h p.t. with pCI-neo (Vector), pCI-M3, both pCI-M3 and pCI-S3, pCI-M3(41-721), or both pCI-M3(41-721) and pCI-S3, CV-1 cells were lysed in nondenaturing buffer and immunoprecipitated (IP) using µNS-specific rabbit polyclonal antiserum (8) (left). In parallel, CV-1 cells transfected with both pCI-M3 and pCI-S3 or both pCI-M3(41-721) and pCI-S3 were lysed in nondenaturing buffer and immunoprecipitated with NS MAb (right). Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using µNS antiserum followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.

Associations with NS and µ2 require different regions of the µNS N terminus.

View larger version (77K): [in this window] [in a new window]   FIG. 5. Association of NS and µ2 requires different regions of the µNS N terminus. IF microscopy of CV-1 cells at 18 h p.t. with both pCI-M3(14-721) and pCI-S3 (top row) or both pCI-M3(14-721) and pCI-M1(T1L) (bottom row) is shown. NS was immunostained with NS-specific mouse MAb 3E10 (5) followed by Alexa 488-conjugated anti-mouse IgG (top left). µ2 was immunostained with µ2-specific rabbit polyclonal antiserum (9) followed by Alexa 488-conjugated anti-rabbit IgG (bottom left). µNS was immunostained with Texas Red-conjugated µNS-specific polyclonal antibodies (9) (right column). Scale bars, 10 µm.

Deletion of NS residues 1 to 11 reduces NS association with µNS.

View larger version (64K): [in this window] [in a new window]   FIG. 6. NS residues 1 to 11 are needed for maximal association with µNS. (A) IF microscopy of CV-1 cells at 18 h p.t. with pCI-S3 (top left), pCI-S3(12-366) (top right), or both pCI-S3(12-366) and pCI-M3 (bottom). NS and NS(12-366) were immunostained with NS-specific mouse MAb 3E10 (5) followed by Alexa 488-conjugated anti-mouse IgG (top left and right and bottom left). µNS was immunostained with Texas Red-conjugated µNS-specific polyclonal antibodies (9) (bottom right). Scale bars, 10 µm. (B) Co-IP of µNS with NS or NS(12-366). At 18 h p.t. with pCI-S3, both pCI-S3 and pCI-M3, pCI-S3(12-366), or both pCI-S3(12-366) and pCI-M3, CV-1 cells were lysed in nondenaturing buffer and immunoprecipitated (IP) with NS MAb. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using µNS-specific rabbit polyclonal antiserum (8) followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.

NS is complexed with both RNA and µNS in infected cells.

View larger version (15K): [in this window] [in a new window]   FIG. 7. NS is complexed with both RNA and µNS in infected cells. (A) Mock- or T1L-infected CV-1 cells were lysed in nondenaturing buffer at 18 h p.i. and immunoprecipitated (IP) using µNS-specific rabbit polyclonal antiserum (8). IP with beads alone (BA) was used as a nonspecific-binding control. The immunoprecipitated proteins were split into two samples that were either treated or not treated with 10 U of RNase ONE (Promega). The samples were then resubjected to centrifugation, and the pellets (left and right) and supernatants (Sup) (middle) were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted using NS-specific rabbit polyclonal antiserum (16) followed by HRP-conjugated anti-rabbit IgG (left and middle) or using µNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence.

NS is complexed with both RNA and µNS in transfected cells.

View larger version (18K): [in this window] [in a new window]   FIG. 8. NS is complexed with both RNA and µNS in transfected cells. CV-1 cells transfected with pCI-neo (Vector), pCI-S3, or both pCI-M3 and pCI-S3 were lysed in nondenaturing buffer at 18 h p.t. and immunoprecipitated (IP) using µNS-specific rabbit polyclonal antiserum (8). The immunoprecipitated proteins were split into two samples, which were either treated or not treated with 10 U of RNase ONE (Promega). The samples were then resubjected to centrifugation, and the pellets (left and right) and supernatants (Sup) (middle) were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted using NS-specific mouse MAb 3E10 (5) followed by HRP-conjugated anti-mouse IgG (left and middle) or µNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Role of NS in reovirus factory formation.

NS may nonetheless play a regulatory role in factory formation (25a). While NS coexpression with µNS did not alter the shape or localization of µNS inclusions, it often resulted in many smaller inclusions that were juxtaposed in the cytoplasm (Fig. 2A). A possible explanation is that association of NS with µNS interferes with µNS-µNS interactions and consequently prevents smaller µNS inclusions from merging to form larger ones. Whether this has any implications for the roles of NS (and µNS) in infection remains to be determined. NS may also play a role in recruiting RNA to the factories (see below).

Previous results suggesting a defect in factory formation by the tsE320 mutant can probably be explained by this strain's defect in protein production (5). Our laboratory has previously reported that the size of the µNS inclusions is dependent on the amount of protein expressed (9); thus, a defect in protein production would likely lead to reduced formation of larger factories. When we introduced the tsE320 mutation into the NS expression plasmid [pCI-S3(M260T)], we found that in most transfected cells it formed large aggregates at restrictive temperature, typical of misfolded proteins (data not shown). Although this mutant NS did not localize to µNS inclusions when coexpressed with µNS at restrictive temperature (data not shown), we think this was likely because the protein was misfolded and nonfunctional in many respects and not because of a specific defect in µNS association. At permissive temperature, localization of NS(tsE320) in the absence or presence of µNS was similar to that of wild-type NS (data not shown). It remains to be determined which specific functions of NS are responsible for the defects in protein and viral dsRNA production exhibited by the tsE320 mutant (10, 14).

NS association with the N terminus of µNS. A previous study has shown that µNS residues 1 to 40 or 41 are both necessary and sufficient for association with µ2 (28). We showed in this study that the N-terminal 40 amino acids of µNS were also necessary for association with NS (Fig. 4). Additionally, we dissected this region of µNS into two smaller regions by showing that deletion of µNS residues 1 to 13 resulted in loss of NS association with µNS but had no effect on µ2 association with µNS (Fig. 5). These findings are especially interesting because a second form of µNS, called µNSC, thought to lack 40 residues from the N terminus of µNS is expressed during infection (24, 38). It is not known if µNSC performs the same functions as µNS or has some distinct role(s). The findings that µNS can associate strongly with both NS and the viral microtubule-associated protein µ2 (9), whereas µNS(41-721) can associate with neither, support the hypothesis that µNS and µNSC are involved in some different processes. Like µNS, µNS(41-721) forms factory-like inclusions in transfected cells (9) and also binds viral core particles in vitro (T. J. Broering, P. L. Joyce, and M. L. Nibert, unpublished data). Based on these findings, we propose that the production of both µNS and µNSC might be needed as part of a regulatory mechanism to balance RNA assortment, minus-strand synthesis, and core assembly within viral factories. In particular, µNS, which can associate with both NS and µ2, might promote RNA-related processes, whereas µNSC, which can associate with neither NS nor µ2 but can still interact with one or more of the core surface proteins (Broering et al., unpublished), might promote capsid assembly-related processes.

Even though we defined two distinct regions of the µNS N terminus required for association with NS and µ2, we have not yet determined if the same molecule of µNS can associate with both proteins simultaneously or if instead NS and µ2 must associate with different molecules of µNS. µNS may act to recruit proteins to viral factories such that they can perform their roles in RNA assortment, minus-strand synthesis, and core assembly. In addition, µNS, by associating with several proteins at once, may act to bring these proteins (e.g., NS and µ2) into close proximity and in particular orientations to perform specific functions within the factories. We have also not yet demonstrated that a region of the µNS N terminus is sufficient for association with NS.

Complexes containing µNS, NS, and RNA. NS is an ssRNA-binding protein that is isolated from infected cells in large RNA-containing complexes (16, 18, 22). The N-terminal 11 residues of NS have been previously shown to be required for both optimal ssRNA binding and formation of the large NS-RNA complexes (16). We showed in this study that deletion of NS residues 1 to 11 diminished, but did not eliminate, the capacity of NS to associate with µNS (Fig. 6). These results suggest that RNA contributes to the NS association with µNS. We further addressed the role of RNA by examining the effect of RNase treatment on NS and µNS association in immunoprecipitated complexes from either infected (Fig. 7) or transfected (Fig. 8) cells. Those studies showed that while a large portion of NS was released from µNS when treated with RNase ONE, a smaller portion of NS remained associated with µNS even following this treatment. This finding suggests that some NS might associate with µNS independently of RNA. In any case, much of the NS associated with µNS is in the form of NS-RNA complexes in both infected and transfected cells. Whether RNA binding alters the conformation of NS to enhance µNS association remains unknown; however, the diminished levels of µNS association with NS(12-366) might indicate that there is a higher-affinity or -avidity interaction between NS and µNS when NS is complexed with RNA.

µNS, NS, and the structural protein 3 were previously shown to be coimmunoprecipitated from infected cells in complexes that contain viral ssRNA (2). While there is strong evidence that NS is an ssRNA-binding protein (17, 18, 22) and 3 is a dsRNA-binding protein (22, 32), it is not known if µNS binds either ss- or dsRNA. Our results showed that NS associated with µNS in viral factories as soon as either protein was detectable in infected cells (Fig. 1) and that, when µNS immunoprecipitates were treated with RNase ONE, a large portion of NS was released into the supernatant while only a smaller portion of NS remained associated with µNS on the beads (Fig. 7). This raises the possibility that the RNA immunoprecipitated using a µNS-specific MAb in the previous studies (2) was not bound to µNS but was instead bound to the associated NS. The RNA-binding properties of µNS are currently under investigation.

The nature of the RNA coimmunoprecipitated with NS and µNS in our experiments remains unknown. NS binds RNA in a sequence-independent manner in vitro (17, 30), and in the present studies it was bound to RNA in cells in the absence of infection (Fig. 8). It is possible that the bound RNA was derived from the NS and µNS expression vectors, but this has not been tested. Previous evidence that ssRNA derived from each of the 10 genome segments can be coimmunoprecipitated with NS, µNS, and 3 from infected-cell lysates (2) has been used to suggest that these proteins cooperate in forming early replication complexes. Our results add to this hypothesis by suggesting that the complexes containing RNA, NS, and µNS are localized to viral factories through either direct or indirect association of NS with the N terminus of µNS. We did not examine the localization of 3 in this study.

Overview: specific protein-protein associations in reovirus factories. Our recent and ongoing studies are identifying a series of specific protein-protein associations involved in forming, and recruiting proteins and RNA to, the viral factories in reovirus-infected cells (Fig. 9). To date, we have identified the 80-kDa nonstructural protein µNS as a key player in these associations. µ2 and NS, the latter in the form of RNA-containing complexes, specifically associate with µNS through a mechanism requiring the µNS N terminus (28). Other evidence suggests that one or more of the surface proteins in viral cores (1, 2, or 2) associates with the more-carboxyl-terminal regions of µNS contained in µNSC (38) (Broering et al., unpublished). Our current hypothesis is that µNS and µNSC are further required during reovirus infection for specifically organizing the viral and cellular factors involved in RNA recruitment and assortment, minus-strand synthesis, and core assembly within the factories.

View larger version (42K): [in this window] [in a new window]   FIG. 9. Model of reovirus protein associations and factory assembly. (A) With most reovirus strains, µ2 associates with cellular microtubules and anchors viral factories to them through association with the µNS N terminus (9, 28). (B) NS, complexed with RNA, is also recruited to viral factories by association with the N terminus of µNS (this study). NS may associate with a µNS molecule before joining the factory (top path) or, alternatively, it might bind to µNS already within the factory (bottom path). (C) µNSC does not bind NS (this study) or µ2 (9), but it does form globular inclusions (9) and does associate with core surface proteins (Broering et al., unpublished), suggesting it is involved in some distinct steps in the replication cycle.

	ACKNOWLEDGMENTS

 
We thank Michelle Becker, Terry Dermody, and coworkers for providing the NS-specific MAb used in these studies. We also thank Jonghwa Kim for sharing his µ2 antiserum, Caroline Piggott for performing antibody titrations, Elaine Freimont and Jason Dinoso for laboratory maintenance and technical assistance, and other members of our laboratory for helpful discussions.

This work was supported by NIH grants RO1 AI47904 (to M.L.N.) and K08 AI52209 (to J.S.L.P) and a junior faculty research grant from the Giovanni Armenise—Harvard Foundation (to M.L.N.). C.L.M. and T.J.B. received additional, respective, support from NIH grant T32 AI07061 to the Combined Infectious Diseases Training Program at Harvard Medical School and NIH grant T32 AI07245 to the Viral Infectivity Training Program at Harvard Medical School.

	FOOTNOTES

 
* Corresponding author. Mailing address: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 645-3680. Fax: (617) 738-7664. E-mail: mnibert{at}hms.harvard.edu.

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