Mammalian Reovirus Nonstructural Protein μNS ... - Journal of Virology

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Feb 28, 2002 - ology and Molecular Genetics, Harvard Medical School, 200 Long- wood Ave., Boston, MA ... ular factories, does not cause increased MT stability or bun- dling. ...... 109:76–86. 15. Heath, C. M., M. Windsor, and T. Wileman.
JOURNAL OF VIROLOGY, Aug. 2002, p. 8285–8297 0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.16.8285–8297.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 76, No. 16

Mammalian Reovirus Nonstructural Protein ␮NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ␮2 in Transfected Cells Teresa J. Broering,1,2 John S. L. Parker,1 Patricia L. Joyce,2† Jonghwa Kim,1,2 and Max L. Nibert1* Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,1 and Department of Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin 537062 Received 28 February 2002/Accepted 9 May 2002

Cells infected with mammalian orthoreoviruses contain large cytoplasmic phase-dense inclusions believed to be the sites of viral replication and assembly, but the morphogenesis, structure, and specific functions of these “viral factories” are poorly understood. Using immunofluorescence microscopy, we found that reovirus nonstructural protein ␮NS expressed in transfected cells forms inclusions that resemble the globular viral factories formed in cells infected with reovirus strain type 3 Dearing from our laboratory (T3DN). In the transfected cells, the formation of ␮NS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller ␮NS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of ␮NS and reovirus protein ␮2 from a different strain, type 1 Lang (T1L), which forms filamentous viral factories, altered the distributions of both proteins. In cotransfected cells, the two proteins colocalized in thick filamentous structures. After nocodazole treatment, many small dispersed globular inclusions containing ␮NS and ␮2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the ␮2 protein from T3DN also colocalized with ␮NS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing ␮NS and ␮2 protein from T3DN and the filamentous structures containing ␮NS and ␮2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirusinfected cells, which is determined by the ␮2-encoding M1 genome segment. We found that the first 40 amino acids of ␮NS are required for colocalization with ␮2 but not for inclusion formation. Similarly, a fusion of ␮NS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the ␮2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between ␮NS and ␮NSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of ␮NS. The capacity of ␮NS to form inclusions and to colocalize with ␮2 in transfected cells suggests a key role for ␮NS in forming viral factories in reovirus-infected cells. sions at 48 h postinfection (p.i.) (33). The inclusions do not contain ribosomes (31, 33) and are not membrane bound (21). To differentiate these inclusions from the inclusion bodies formed by the aggregation of misfolded proteins (19), we refer to the reovirus phase-dense inclusions as “viral factories.” The viral and cellular factors responsible for the formation and morphology of viral factories and the exact functions and effects of these factories during reovirus infection are largely unknown. A reovirus strain-dependent difference in the kinetics of viral factory formation was recently mapped to the M1 genome segment, which encodes the structurally minor core protein ␮2, and secondarily to the S3 genome segment, which encodes the nonstructural protein ␴NS (22). Another recent study used temperature-sensitive reovirus mutants to define roles in viral factory formation for both ␮2 and ␴NS (2). It was suggested that viral RNA-protein complexes containing ␴NS nucleate inclusion formation and that other viral proteins, including ␮2, are recruited to these complexes. However, the expression of ␴NS in the absence of other viral proteins does not lead to factory formation; instead, ␴NS is diffusely distributed within the cytoplasm and nucleus (M. M. Becker, T. R. Peters, and

The replication and assembly of viruses are often concentrated in specific locations within infected cells, such as on the actin cytoskeleton for human parainfluenza virus type 3 (13), on the outer mitochondrial membranes for flock house virus (24), in cytoplasmic inclusions for vaccinia virus (39), and in nuclear inclusions for herpes simplex virus (32). The nonfusogenic mammalian orthoreoviruses (reoviruses) are believed to replicate and assemble in cytoplasmic phase-dense inclusions in infected cells (31). These inclusions contain viral doublestranded RNA (34), viral proteins (9, 31), partially and fully assembled viral particles (31), and both microtubules (MTs) and thinner “kinky” filaments suggested to be intermediate filaments (8, 9, 35). It was reported that the intermediate filament vimentin is rearranged in reovirus-infected CV-1 cells such that it surrounds and is incorporated into the large inclu* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-4828. Fax: (617) 738-7664. E-mail: [email protected]. † Present address: Department of Radiation Oncology, University of North Carolina—Chapel Hill, Chapel Hill, NC 27599. 8285

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T. S. Dermody, Abstr. 20th Meet. Am. Soc. Virol., abstr. W28-7, 2001; C. L. Miller, T. J. Broering, J. S. L. Parker, and M. L. Nibert, unpublished data). Thus, although ␴NS may be required, it is not sufficient for viral factory formation, and other viral components are needed for factory morphogenesis. Parker et al. recently described two strain-dependent reovirus factory morphologies: filamentous, induced by the majority of strains tested, and globular, induced by only two strains (28). The filamentous viral factories are colinear with MTs, and MTs are stabilized and bundled in cells infected with strains that form filamentous factories, including reovirus strain type 1 Lang (T1L). In contrast, infection with reovirus strain type 3 Dearing from our laboratory (T3DN), a strain that forms globular factories, does not cause increased MT stability or bundling. Strain-dependent differences in both viral factory morphology and MT stabilization are determined by the ␮2encoding M1 genome segment (28). When ␮2 derived from T1L [␮2(T1L)] is expressed in cells, it colocalizes with MTs and causes MT bundling and stabilization, whereas ␮2 derived from T3DN [␮2(T3DN)] does not (28). These findings implicate protein ␮2 as a major determinant of viral factory association with MTs. However, ␮2 does not form structures that resemble factories when expressed alone, suggesting that one or more other viral proteins are involved in forming the matrix of the factories. A reovirus nonstructural protein encoded by the M3 genome segment, ␮NS, can bind to a reovirus subviral particle (core) in vitro and form large amorphous complexes (5). Negative-stain electron microscopy (EM) revealed an extensive matrix surrounding the core particles; the morphology of this ␮NS and core complex resembled the morphology of factories in thin sections of reovirus-infected cells viewed by EM (33). By immunofluorescence (IF) microscopy, ␮NS was found concentrated within viral factories in infected cells (25, 28). Based on this evidence, we hypothesized as part of the current study that ␮NS plays a major role in viral factory structure and morphogenesis in infected cells. ␮NS is an 80-kDa protein expressed at high levels in infected cells (41, 42). A second form of the protein, called ␮NSC, is also produced (20). Chemical cleavage of ␮NS and ␮NSC from infected cells demonstrated that ␮NSC lacks about 5 kDa of sequence from the amino (N) terminus of ␮NS (41). It was hypothesized that the synthesis of ␮NS results from ribosome initiation at the first AUG codon in the M3 plus-strand RNA (nucleotides 19 to 21) whereas the synthesis of ␮NSC results from initiation at the second, in-frame, AUG codon (nucleotides 139 to 141) (41). These first and second AUG codons are conserved in the three reovirus strains for which M3 sequences have been determined to date (23). The kinetics of ␮NSC formation in infected cells indicate that ␮NS and ␮NSC do not have a precursor-product relationship (41), supporting the hypothesis that ␮NSC arises by secondary initiation rather than by cleavage of full-length ␮NS. The relative functions of ␮NS and ␮NSC in infected cells are not known. To address the hypothesis that ␮NS and ␮NSC play roles in viral factory formation, we expressed these proteins in the absence of other reovirus proteins by transfection of DNA expression vectors containing the encoding genes. The results suggest a role for these proteins in factory formation. Moreover, colocalization and redistribution of ␮NS and ␮2 when coexpressed suggest

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that specific associations between these two proteins may be key to forming viral factories and recruiting the factors necessary for replication and assembly. MATERIALS AND METHODS Cells, viruses, and reagents. CHO, Mv1Lu, and CV-1 cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Rockville, Md.) containing 10% fetal bovine serum (HyClone Laboratories, Logan, Utah) and 10 ␮g of gentamicin (Life Technologies) per ml. Reovirus strains T1L and T3DN were laboratory stocks. The designation T3DN differentiates our laboratory strain from another strain (T3DC) that differs in inclusion phenotype and M1 sequence as described previously (28). All enzymes were obtained from New England Biolabs, Inc. (Beverly, Mass.), unless otherwise stated. Antibodies. Monoclonal antibodies to Cy3-conjugated ␤-tubulin (TUB 2.1) were obtained from Sigma (St. Louis, Mo.). Goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG conjugated to Alexa 488 or Alexa 594 were obtained from Molecular Probes (Eugene, Oreg.). Monoclonal antibodies to green fluorescent protein (GFP) (JL-8) were obtained from Clontech (Palo Alto, Calif.). We used rabbit polyclonal antisera against ␮NS (5) and ␮2 (described below). ␮NS and ␮2 polyclonal IgG antibodies purified with protein A-Sepharose were directly conjugated to Texas red and Oregon green by following the manufacturer’s procedure (Molecular Probes). As determined by IF analysis, the ␮NS antiserum did not stain cells expressing ␮2 or GFP, and the GFP antibodies did not stain cells expressing ␮NS or ␮2. The ␮2 antiserum showed a low level of background nuclear fluorescence with paraformaldehyde (PFA) fixation that was reduced with methanol fixation as previously described (28), but the antiserum did not stain ␮NS or GFP in transfected cells. Rabbit polyclonal antiserum specific for ␮2 was produced by using Escherichia coli-expressed protein. The T1L M1 gene was excised from pBluescript II KS(⫹) (Stratagene, La Jolla, Calif.) (28) with SmaI and XhoI and ligated to pET-21b (Novagen, Madison, Wis.) cut with HindIII and XhoI, with the HindIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). ␮2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen). In brief, expression was induced with 1 mM isopropyl-␤-D-thiogalactopyranoside. The cells were incubated at 37°C for 3 h, pelleted, resuspended, and lysed by sonication. Approximately 50% of the insoluble fraction was ␮2. To further purify ␮2 from the insoluble fraction, it was subjected to preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electroeluted (14). The eluent was concentrated in dialysis tubing by buffer absorption with polyethylene glycol. The antiserum was generated in a rabbit by the Polyclonal Antibody Service at the Animal Care Unit of the University of Wisconsin Medical School. Rat antiserum specific for the N-terminal 41 amino acids of ␮NS was produced by using a fusion protein of glutathione S-transferase (GST) and amino acids 1 to 41 of the T1L ␮NS protein [GST-␮NS(T1L)(1–41)]. To express the protein, pGEM4Z-M3(T1L) (5) was cut with AflIII and SalI, and the piece of DNA containing M3 nucleotides 1 to 142 was isolated. The Klenow fragment was used to convert the AflIII site to a blunt end. The small piece of DNA was ligated to pGEX-4T-3 (Amersham Biosciences, Piscataway, N.J.) cut with NotI and SalI, with the NotI site converted to a blunt end with the Klenow fragment; this procedure generated pGEX-M3(T1L)(1–41). The fusion protein, GST␮NS(T1L)(1–41), was expressed in BL21-DE3 cells and purified by using glutathione-agarose beads (Pierce, Rockford, Ill.) according to the instructions in the pGEX manual (Amersham Biosciences). The antiserum was generated in a rat by the Polyclonal Antibody Service at the Animal Care Unit of the University of Wisconsin Medical School. Generation of ␮NS mammalian expression constructs. The DNA clone of the T3D M3 gene used in this study was originally generated by Cashdollar et al. (7). As a result, it was likely derived from a viral plaque isolate most closely related to T3DC (28). We have chosen not to include the Cashdollar (C) or Nibert (N) designation on the M3 gene (28) because there is as yet no demonstrated sequence or functional difference between the M3 genes of the T3DC and T3DN viruses. The T3D M3 gene was cut from pGEM4Z-M3(T3D) (5) by using the KpnI and SalI sites. T4 DNA polymerase was used to convert the KpnI site to a blunt end, which was ligated to pCI-neo (Promega, Madison, Wis.) that had been cut with SmaI and XhoI; this procedure generated pCI-M3(T3D). The T1L M3 gene was cut from pGEM4Z-M3(T1L) by using the BamHI and NheI sites. The Klenow fragment was used to convert the BamHI site to a blunt end, which was ligated to pCI-neo that had been cut with SmaI and NheI; this procedure generated pCI-M3(T1L).

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Generation of a ␮NS(41–721) expression construct. To generate a vector to direct the expression of a ␮NS protein that lacks N-terminal amino acids, we exploited the second in-frame AUG codon in the T1L M3 gene (nucleotides 139 to 141) and a unique StyI restriction site in the T1L M3 gene (nucleotides 62 to 67). We used the StyI restriction site to introduce a frameshift into the ␮NS open reading frame between the first two AUG codons. pGEM4Z-M3(T1L) was cut with StyI, and the ends were made blunt with the Klenow fragment. The plasmid was religated, thereby adding an insertion of 4 bp after nucleotide 62. The mutated M3 gene was cloned into pCI-neo as described above for the T1L M3 gene, generating pCI-M3(41–721). The insertion shifts the reading frame from the first AUG, associating it with a stop codon at nucleotides 79 to 81. Ribosome initiation at the first AUG codon (nucleotides 19 to 21) should therefore produce a protein containing only amino acids 1 to 16 of ␮NS and an additional 4 amino acids. We have not been able to detect this product in immunoblots with ␮NS antisera (data not shown). Ribosome initiation at the second AUG codon should produce a protein containing amino acids 41 to 721 of ␮NS, with a predicted molecular mass of 76 kDa. We have detected this product (see Fig. 7). Generation of GFP fusion vectors. To generate a fusion of ␮NS amino acids 1 to 41 to the N terminus of GFP, pGEM4Z-M3(T1L) was cut with AflIII and NheI, and the piece of DNA containing M3 nucleotides 1 to 142 was isolated. The Klenow fragment was used to convert the AflIII site to a blunt end, which was ligated to pEGFP-N1 (Clontech) that had been cut with BamHI and NheI, with the BamHI site converted to a blunt end with the Klenow fragment; this procedure generated pEGFP-M3(1–41). To generate a full-length ␮NS fusion with GFP, the stop codon of the M3 gene was removed by using PCR with a primer complementary to the 5⬘ end of M3 containing no stop codon and a new BamHI site. Nucleotides 1842 to 2181 of M3 were amplified with a forward primer complementary to nucleotides 1842 to 1859, a reverse primer (5⬘-GTGTATCCGCCAGCTCATCAGTTGGAAC-3⬘), pGEM4Z-M3(T1L) as a template, and Vent polymerase. The PCR product was cut with SalI and BamHI and ligated to pGEM4Z-M3(T1L) that had been cut with SalI and BamHI to remove nucleotides 1842 to 2241; this procedure generated pGEM4Z-M3(T1L)(no stop). The intended mutation was verified by sequencing. The M3 gene was cut from pGEM4Z-M3(T1L)(no stop) with NheI and BamHI and ligated to pEGFP-N1 that had been cut with NheI and BamHI; this procedure generated pEGFP-M3. IF microscopy. CV-1 cells were seeded on the day before transfection or infection at a density of 1.0 ⫻ 104 per cm2 in six-well plates (9.6 cm2 per well) containing glass coverslips. Transfections were performed with 2 ␮g of DNA (total) and 6 ␮l of Lipofectamine (Life Technologies) per well according to the manufacturer’s directions. Infections were done for 1 h at room temperature with 200 ␮l of phosphate-buffered saline (PBS) (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4 [pH 7.5]) supplemented to contain 2 mM MgCl2. Cells were further incubated at 37°C before being processed for IF microscopy. Cells were fixed for 10 min at room temperature in 2% PFA or for 3 min at ⫺20°C in 100% methanol. No significant differences in morphology were seen between PFA fixation and methanol fixation of inclusions or filamentous structures in infected or transfected cells. PFA fixation was insufficient to fix GFP and the GFP fusion to ␮NS amino acids 1 to 41. Instead, methanol fixation was used, and GFP was recognized with the specific monoclonal antibody. Cells were permeabilized, blocked, incubated with antibodies and 4⬘,6-diamidino-2-phenylindole (DAPI), and mounted as described previously (28). Samples were examined by using a Nikon TE-300 inverted microscope equipped with phase and fluorescence optics, and images were collected digitally as described previously (28). Images were processed and prepared for presentation by using Photoshop and Illustrator software (Adobe Systems, San Jose, Calif.). Immunoblot analysis. To compare viral proteins expressed in infected and transfected cells, we collected cell lysates at 18 to 24 h p.i. or posttransfection (p.t.). CV-1 cells (1.2 ⫻ 106) were washed briefly in PBS, scraped into 2 ml of PBS, and pelleted. The pelleted cells were resuspended in 60 ␮l of PBS containing protease inhibitors (Roche, Indianapolis, Ind.), lysed in sample buffer, boiled for 10 min, and subjected to SDS-PAGE. Proteins were electroblotted from the gels to nitrocellulose in 25 mM Tris–192 mM glycine (pH 8.3). The binding of antibodies was detected with alkaline phosphatase-coupled goat anti-mouse or anti-rabbit (Bio-Rad Laboratories, Hercules, Calif.) or anti-rat (Pierce) IgG and the colorimetric reagents p-nitroblue tetrazolium chloride and 5-bromo-4chloro-3-indolylphosphate p-toluidine salt (Bio-Rad).

RESULTS Globular inclusions are formed in cells that express ␮NS.

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To test whether ␮NS can induce structures that resemble viral factories when expressed in the absence of other reovirus proteins, we transfected CV-1 cells with expression vectors containing the M3 gene from either reovirus T1L [pCI-M3(T1L)] or reovirus T3D [pCI-M3(T3D)] and examined the subcellular distribution of ␮NS by IF microscopy at 18 h p.t. Large and small globular phase-dense inclusions, with smooth edges, were observed in the cytoplasm of cells transfected with either M3 gene (Fig. 1A). These structures contained ␮NS and closely resembled the ␮NS-containing globular phase-dense viral factories in reovirus T3DN-infected cells (28) (Fig. 1A). The untransfected cells in the samples (Fig. 1A, top panels) and separate untransfected cell controls (data not shown) did not react with the ␮NS-specific antiserum and did not contain globular phase-dense structures. We observed a similar distribution of the ␮NS protein from T1L [␮NS(T1L)] in transfected CHO and Mv1Lu cells (supplemental data can be found at http://micro.med.harvard.edu/nibert/suppl/broering02a/ fig1.html). Unlike the filamentous viral factories in T1L-infected cells (28), the inclusions in cells expressing ␮NS(T1L) were globular (Fig. 1A). Analysis of cells at different times revealed that at 6 h p.t., ␮NS inclusions were uniformly small and spread throughout the cytoplasm (Fig. 1B), whereas at 18 h p.t. (Fig. 1A) and 36 h p.t. (Fig. 1B), large perinuclear inclusions were present along with smaller inclusions. These changes in the size and distribution of ␮NS inclusions over time p.t. resemble the timedependent changes in the viral factories in T3DN-infected cells (2, 28). The striking similarity between the viral factories in T3DN-infected cells and the inclusions in cells transfected with either the T1L or the T3D M3 gene is consistent with our hypothesis that ␮NS plays a role in the formation and structure of the viral factories in infected cells. Because misfolded proteins can accumulate in globular phase-dense structures called aggresomes (18), which appear similar to some viral inclusions (15, 28), we examined samples for two hallmarks of aggresome formation in association with the ␮NS inclusions: polyubiquitination and collapse of the vimentin intermediate filament network around the inclusions (18). For CV-1 cells transfected with pCI-M3(T1L), we found no staining of the ␮NS inclusions with an antibody to polyubiquitin and no evidence for redistribution of vimentin intermediate filaments to surround the inclusions (supplemental data can be found at http://micro.med.harvard.edu/nibert/ suppl/broering02a/fig2.html), suggesting that the ␮NS inclusions are not aggresomes. Size and distribution of ␮NS(T1L) globular inclusions are dependent on MTs. Based on the previous observations that MTs are embedded in reovirus factories (8, 12, 35) and that ␮NS may associate with MTs (25), we examined samples for the colocalization of ␮NS(T1L) with MTs by staining pCIM3(T1L)-transfected cells for ␮NS and ␤-tubulin. ␮NS globular inclusions did not colocalize with or reorganize MTs (Fig. 2), but small isolated inclusions on the periphery of the cells were found adjacent to MTs (Fig. 2, inset). These findings are similar to those for the viral factories in T3DN-infected cells (28). To determine whether the morphology of ␮NS globular inclusions is altered by the depolymerization of MTs, we treated transfected cells with 10 ␮M nocodazole for 12 h be-

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FIG. 1. Distribution of ␮NS in transfected and reovirus-infected cells. (A) Phase-contrast microscopy (left column) and IF microscopy (right column) of CV-1 cells transfected (upper four panels) with 2 ␮g of pCIM3(T1L) or pCI-M3(T3D) per well or infected (lower four panels) with T3DN or T1L at a multiplicity of infection of 5. The cells were fixed at 18 h p.t. or p.i. and immunostained with rabbit anti-␮NS IgG directly conjugated to Texas red (T3DN and T1L infection) or Oregon green [pCIM3(T3D) transfection] or immunostained with rabbit anti-␮NS serum followed by goat anti-rabbit IgG conjugated to Alexa 488 [pCI-M3(T1L) transfection]. (B) CV-1 cells transfected with 2 ␮g of pCI-M3(T1L) per well were fixed at 6 h p.t. (left) or 36 h p.t. (right) and immunostained with Oregon green-conjugated anti-␮NS rabbit IgG. Bars, 10 ␮m.

ginning at 6 h p.t. to depolymerize MTs as described previously (28). With nocodazole treatment at 6 h p.t., MTs were completely depolymerized, and ␮NS(T1L) was located in numerous small globular inclusions dispersed throughout the cyto-

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plasm (Fig. 2). When transfected cells were treated with 10 ␮M nocodazole for only 1 h beginning at 17 h p.t., MTs were completely depolymerized, but the large perinuclear ␮NS inclusions were not disrupted (Fig. 2). Similar observations were made for the viral factories in T3DN-infected cells (28). We conclude that the MT network is required for the formation of large perinuclear ␮NS inclusions but is not required for the maintenance of the large inclusions once they are formed. ␮NS and ␮2 colocalize when coexpressed in cells. The ␮NS globular inclusions in transfected cells expressing ␮NS(T1L) contrast with the filamentous distribution of ␮NS in T1L-infected cells (28) (Fig. 1A, compare top and bottom panels). Because the filamentous appearance of viral factories in T1Linfected cells is determined by the capacity of ␮2(T1L) to associate with MTs (28), we hypothesized that ␮2 associates with ␮NS and redistributes it to MTs. To test this hypothesis, we transfected CV-1 cells with pCI-M3(T1L) and pCIM1(T1L) at a 1:1 (wt/wt) ratio and examined the distribution of ␮NS and ␮2 by using protein-specific polyclonal antisera. The ␮NS(T1L) and ␮2(T1L) proteins colocalized in thick filamentous structures that surrounded the nucleus (Fig. 3A). The distribution of ␮NS(T1L) and ␮2(T1L) in cells coexpressing the two proteins was clearly different from the distribution of each protein expressed separately (compare Fig. 3A with Fig. 1A and 3B). The results suggest that ␮NS(T1L) and ␮2(T1L) associate and that ␮2(T1L) likely mediates the redistribution of ␮NS(T1L) to MTs. When expressed in transfected cells, the ␮2(T3DN) protein does not have a filamentous distribution and does not colocalize with MTs (28). Instead, ␮2(T3DN) is diffusely distributed throughout the cytoplasm and nucleus or, in cells that express more of the protein, is concentrated in rough-edged structures that differ in appearance from ␮NS inclusions and may be aggregates of misfolded protein (28) (Fig. 3B). To determine whether the lack of MT association would affect the capacity of ␮2(T3DN) to colocalize with ␮NS(T1L), CV-1 cells were cotransfected with pCI-M3(T1L) and pCI-M1(T3DN) at a 1:1 (wt/wt) ratio. We found that the distribution of the ␮2(T3DN) protein changed when it was coexpressed with ␮NS(T1L) such that the two proteins were colocalized in large globular inclusions with smooth edges (Fig. 3A), very similar to those seen when ␮NS(T1L) was expressed in the absence of ␮2 (Fig. 1A). Results essentially the same as those described for ␮NS(T1L) were observed when the ␮NS protein from T3D [␮NS(T3D)] was coexpressed with either ␮2(T1L) or ␮2(T3DN) (data not shown). These results indicate that the colocalization of ␮NS and ␮2 is independent of the strain derivations of these proteins but that the morphology of ␮NS/␮2 structures is determined by the strain-dependent difference in the association of ␮2 with MTs. As described previously, some of the ␮2 protein—especially ␮2(T1L)—appears to be located in the nuclei of many transfected cells (28) and may be concentrated in as-yet-unidentified nuclear structures (Fig. 3B). When we examined the distribution of ␮2(T1L) or ␮2(T3DN) in cells coexpressing ␮NS(T1L), we found that the nuclear association of ␮2 was much less evident (Fig. 3A). This result suggests the existence of an equilibrium between ␮2 proteins in different subcellular compartments (nucleus and cytoplasm) that is altered by the association of ␮2 with ␮NS.

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FIG. 2. Distribution of ␮NS(T1L) and ␤-tubulin in transfected cells with and without nocodazole treatment. CV-1 cells transfected with 2 ␮g of pCI-M3(T1L) per well were left untreated (upper panels), treated with 10 ␮M nocodazole added at 6 h p.t. (middle panels), or treated with 10 ␮M nocodazole added at 17 h p.t. (lower panels). The cells were fixed at 18 h p.t. and immunostained with Oregon green-conjugated anti-␮NS rabbit IgG (red) (first column) and Cy3-conjugated mouse monoclonal antibody to ␤-tubulin (green) (second column). Nuclei were counterstained with DAPI (blue). The boxed area in the merged image (third column) is enlarged to show detail (inset). Bars, 10 ␮m.

Varying the relative levels of ␮NS and ␮2 expression alters the morphology of ␮NS/␮2 structures. Although the globular inclusions containing ␮NS(T1L) and ␮2(T3DN) closely resemble the globular factories in T3DN-infected cells, the filamentous structures containing ␮NS(T1L) and ␮2(T1L) lack the globular areas connected by thin filaments found in T1L-infected cells (28) (compare Fig. 3A with Fig. 1A). To investigate the effects of changes in the levels of protein expression on the morphology of ␮NS/␮2 structures, we cotransfected cells with pCI-M3(T1L) and either pCI-M1(T1L) or pCI-M1(T3DN) at a ratio of 8:1, 1:1, or 1:8 (wt/wt). We found that in all instances, ␮NS(T1L) colocalized with ␮2 (Fig. 4A); however, the relative amounts of transfected DNA affected the morphology of ␮NS/␮2 structures. When more of the ␮NS expression plasmid was transfected relative to the ␮2 expression plasmid (8:1), globular inclusions connected by thin filaments were predominantly seen with ␮NS(T1L) and ␮2(T1L) and large globular perinuclear inclusions were predominantly seen with ␮NS(T1L) and ␮2(T3DN) (Fig. 4A). Alternatively, when less of the ␮NS expression plasmid was transfected relative to the ␮2 expression plasmid (1:8), the morphology of ␮NS/␮2 structures was closer to that of ␮2 alone: distributed filaments for ␮2(T1L) and small perinuclear structures, with rough edges, for ␮2(T3DN) (Fig. 4A). In addition, the association of ␮2 with

the nucleus was more evident in these cells (Fig. 4A). Structures containing ␮NS and ␮2 in cells transfected with M3 and M1 at a ratio of 8:1 most closely resembled the viral factories in reovirus-infected cells (compare Fig. 4A with Fig. 1A), suggesting that this relative level of protein expression most closely mimics that in infected cells. To analyze the relative levels of ␮NS and ␮2 expression in transfected cells, samples identical to those used for the IF microscopy experiments were analyzed by SDS-PAGE and immunoblotting (Fig. 4B). In both T1L- and T3DN-infected cells, ␮NS was expressed at a higher level than ␮2 (Fig. 4B). In transfected cells, the ratio of M3 DNA to M1 DNA changed the relative levels of ␮NS and ␮2 expression, and as the IF results suggested, an M3/M1 ratio of 8:1 provided relative levels of ␮NS and ␮2 expression that were most similar to those in infected cells (Fig. 4B). It is thus clear that the relative levels of ␮NS and ␮2 expression are important for determining particular aspects of inclusion morphology. ␮NS(T1L) is colinear with MTs in the presence of ␮2(T1L) but not in the presence of ␮2(T3DN). It was previously shown that ␮2(T1L) colocalizes with MTs in transfected cells and that the filamentous inclusions in T1L-infected cells are colinear with MTs (28). Hence, we examined samples for the colocalization of ␮NS/␮2 inclusions with MTs by costaining trans-

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FIG. 3. Colocalization of ␮NS(T1L) and ␮2 in cotransfected cells. (A) CV-1 cells were cotransfected with 1 ␮g of pCI-M3(T1L) and 1 ␮g of pCI-M1(T1L) (upper panels) or 1 ␮g of pCI-M3(T1L) and 1 ␮g of pCI-M1(T3DN) (lower panels) per well and fixed at 18 h p.t. Cells were immunostained with Texas red-conjugated anti-␮NS rabbit IgG (red) (first column) and Oregon green-conjugated ␮2 rabbit IgG (green) (second column). Nuclei were counterstained with DAPI (blue). (B) CV-1 cells were transfected with 2 ␮g of pCI-M1(T1L) (left) or pCI-M1(T3DN) (middle and right) per well, fixed at 18 h p.t., and stained with rabbit anti-␮2 polyclonal serum and goat anti-rabbit IgG conjugated to Alexa 488. Bars, 10 ␮m.

fected cells for ␮NS and ␤-tubulin (Fig. 5). Similar to previous findings for T1L-infected cells (28), we did not find significant colocalization of ␮NS(T1L) with MTs, but we did find that the finer filamentous structures in cells coexpressing ␮NS(T1L) and ␮2(T1L) were colinear with MTs (Fig. 5, upper inset). In cells expressing high levels of recombinant proteins, we detected decreased amounts of MTs stained with the tubulinspecific antibody (supplemental data can be found at http: //micro.med.harvard.edu/nibert/suppl/broering02a/fig3.html), suggesting that the ␮NS(T1L)/␮2(T1L) inclusions mask the underlying MTs and prevent access of the tubulin-specific antibody (28). We also cotransfected cells with pCI-M3(T1L) and pCI-M1(T3DN) and costained samples for ␮NS and ␤-tubulin (Fig. 5). ␮NS globular inclusions did not colocalize with or reorganize MTs and were not colinear with MTs (Fig. 5, lower inset), like the viral factories in T3DN-infected cells (28) and inclusions formed by ␮NS(T1L) expressed in the absence of other viral proteins (Fig. 2). We obtained similar results when cells were cotransfected with ␮NS and ␮2 expression plasmids at a 7:1 ratio (Fig. 5) or a 1:1 M3/M1 antibody ratio (Fig. 5 and supplemental data that can be found at http://micro.med .harvard.edu/nibert/suppl/broering02a/fig3.html). These results

suggest that ␮NS(T1L)/␮2(T1L) inclusions associate with MTs. The morphology of ␮NS/␮2 inclusions depends on an intact MT network. To test whether ␮NS(T1L)/␮2(T1L) filamentous structures are altered by the depolymerization of MTs, we treated cotransfected cells with 10 ␮M nocodazole for 12 h beginning at 6 h p.t. and analyzed the distribution of ␮NS and ␮2 by IF microscopy. Simultaneous control experiments confirmed that MTs were depolymerized by this treatment. In cells coexpressing ␮NS(T1L) and ␮2(T1L), nocodazole treatment at 6 h p.t. prevented the formation of filamentous structures. Instead, ␮NS(T1L) and ␮2(T1L) colocalized in many small globular structures that were dispersed throughout the cytoplasm (Fig. 6). The appearance of small dispersed globular structures after MT depolymerization was also noted for both T3D- and T1L-infected cells treated with nocodazole at 6 h p.i. (28) and for pCI-M3(T1L)-transfected cells treated with nocodazole at 6 h p.t. (Fig. 2). To determine whether MT depolymerization would also affect the morphology of ␮NS(T1L)/ ␮2(T3DN) inclusions, we treated cotransfected cells with 10 ␮M nocodazole for 12 h beginning at 6 h p.t. Cells coexpressing ␮NS(T1L) and ␮2(T3DN) showed a dispersed distribution

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of small globular structures in which ␮NS(T1L) and ␮2(T3DN) colocalized (Fig. 6). We obtained similar results when cells were cotransfected with ␮NS and ␮2 expression plasmids at a 7:1 or a 1:1 M3/M1 ratio (Fig. 6 and data not shown). These results show that intact MTs are required for the formation of filamentous structures in cells expressing ␮NS(T1L) and ␮2(T1L) and for the formation of large perinuclear inclusions in cells expressing ␮NS(T1L) and ␮2(T3DN). When cells coexpressing ␮NS(T1L) and ␮2(T1L) were treated with 10 ␮M nocodazole for 1 h beginning at 17 h p.t. and costained for ␮NS and ␤-tubulin, the ␮NS(T1L)/␮2(T1L) filamentous structures were not disrupted (supplemental data can be found at http://micro.med.harvard.edu/nibert/suppl /broering02a/fig4.html). MTs in untransfected cells were depolymerized by treatment with nocodazole for 1 h, but MTs in cells that contained ␮NS(T1L) and ␮2(T1L) remained intact (supplemental data can be found at http://micro.med.harvard .edu/nibert/suppl/broering02a/fig4.html). MTs in T1L-infected cells were also resistant to depolymerization by 1 h of nocodazole treatment beginning at 17 h p.t. (28), but MTs in transfected cells expressing only ␮NS(T1L) were not resistant (Fig. 2). Parker et al. previously identified the M1 genome segment as the determinant of the viral strain-dependent difference in MT stabilization and demonstrated that MTs are stabilized in transfected cells expressing ␮2(T1L) but not ␮2(T3DN) (28). The current results suggest that ␮2(T1L) also stabilizes MTs when coexpressed with ␮NS. The ␮NS N terminus is required for colocalization with ␮2. A second form of ␮NS, called ␮NSC, is found in reovirusinfected cells and is missing amino acids from the N terminus of ␮NS (41). To determine whether the N terminus of ␮NS is required for inclusion formation and colocalization with ␮2, we engineered a construct [pCI-M3(41–721)] to allow initiation at the second AUG codon in the M3 gene, producing a protein containing amino acids 41 to 721 of ␮NS(T1L) [␮NS(41–721)]. The protein expressed in cells upon transfection of pCIM3(41–721) reacted with the full-length ␮NS polyclonal antiserum but was not recognized by a polyclonal antiserum generated to the first 41 amino acids of ␮NS (Fig. 7A); these results confirm that the N terminus of ␮NS is absent from ␮NS(41–721). Full-length ␮NS expressed in transfected cells and in reovirus-infected cells was recognized by both fulllength and N-terminal ␮NS polyclonal antisera (Fig. 7A).

FIG. 4. Morphologies of ␮NS/␮2 structures with different relative levels of expression of ␮NS(T1L) and ␮2. (A) CV-1 cells were cotransfected with 2 ␮g of DNA (total) of pCI-M3(T1L) and pCI-M1(T1L) (upper six panels) or pCI-M3(T1L) and pCI-M1(T3DN) (lower six panels) with different ratios of M3 DNA to M1 DNA (8:1, 1:1, and 1:8) per well. Cells were fixed at 18 h p.t. and immunostained for ␮NS (left column) and ␮2 (right column) as described in the legend to Fig. 3A. Bars, 10 ␮m. (B) CV-1 cells were transfected with 1 ␮g of pCIM3(T1L), pCI-M1(T1L), or pCI-M1(T3DN) and 1 ␮g of pCI-neo as a carrier plasmid per well and cotransfected as described above. CV-1 cells were infected (inf) with T1L or T3DN at a multiplicity of infection of 5. Lysates were collected at 18 h p.t. or p.i. and analyzed by SDSPAGE and immunoblotting with polyclonal ␮NS and ␮2 antisera (␣␮NS and ␣-␮2, respectively). A band appearing in untransfected CV-1 cells is indicated on the right with an asterisk. The positions of ␮NS and ␮2 are indicated on the right.

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FIG. 5. Distribution of ␮NS(T1L) and ␤-tubulin in cells coexpressing ␮NS(T1L) and ␮2. CV-1 cells were transfected with 1.75 ␮g of pCI-M3(T1L) and 0.25 ␮g of pCI-M1(T1L) (upper panels) or 1.75 ␮g of pCI-M3(T1L) and 0.25 ␮g of pCI-M1(T3DN) (lower panels) per well. Cells were fixed at 18 h p.t. and immunostained for ␮NS (red) and ␤-tubulin (green) as described in the legend to Fig. 2. The boxed areas in the merged images are enlarged to show detail (insets). Colinearity of ␮NS and tubulin is indicated by arrows. Bars, 10 ␮m.

Phase-contrast microscopy of CV-1 cells expressing ␮NS(41– 721) revealed globular phase-dense structures that contained ␮NS(41–721), as determined by IF microscopy (Fig. 7B). Therefore, amino acids 1 to 40 of ␮NS are not required for the formation of globular inclusions in transfected cells. When ␮NS(41–721) was coexpressed with ␮2(T1L), the two proteins did not colocalize in filamentous structures (Fig. 7C). Rather, when coexpressed with ␮2(T1L), ␮NS(41–721) was seen in globular inclusions similar to those seen when the protein was expressed without ␮2 (Fig. 7B), and ␮2(T1L) had a filamentous and nuclear distribution similar to that seen when it was expressed without ␮NS (28) (Fig. 3B). ␮NS(41– 721) and ␮2(T3DN) also failed to colocalize (Fig. 7C). Smoothedged globular structures containing ␮NS(41–721), similar to those found without ␮2 coexpression (Fig. 7B), were surrounded by ␮2(T3DN)-containing rough-edged structures; this appearance was similar to the pattern of ␮2(T3DN) expressed in the absence of other reovirus proteins (28) (Fig. 3B). We conclude that amino acids 1 to 40 of ␮NS(T1L) are required for colocalization with ␮2 but not for inclusion formation. ␮NS(T1L) amino acids 1 to 41 are sufficient for ␮2(T1L) colocalization. To determine whether the N-terminal amino acids of ␮NS that are required for ␮2 colocalization are sufficient for colocalization, we constructed a plasmid [pEGFPM3(1–41)] that expresses amino acids 1 to 41 of ␮NS(T1L) fused to the N terminus of GFP [␮NS(1–41)-GFP]. To control for any effect of GFP on ␮NS/␮2 colocalization or inclusion formation, a plasmid (pEGFP-M3) was constructed that expresses full-length ␮NS(T1L) fused to the N terminus of GFP (␮NS-GFP), and a plasmid expressing GFP alone (pEGFP) was also used. Cells transfected with pEGFP-M3(1–41) and pEGFP-M3 produced proteins of the expected sizes that were recognized by immunoblotting with both full-length ␮NS polyclonal antiserum and GFP antibodies, whereas cells expressing

GFP alone produced a protein that was recognized by only GFP antibodies (Fig. 8A). The N-terminal antiserum generated against amino acids 1 to 41 of ␮NS recognized ␮NS(1– 41)-GFP and ␮NS-GFP but not GFP (data not shown). By immunoblotting, a protein of about the same size as native GFP was detected with the ␮NS(1–41)-GFP fusion protein (Fig. 8A), but the fusion protein was the predominant product (Fig. 8A). The production of this GFP-sized protein could have resulted from removal of the fused, ␮NS(1–41) portion by cleavage or from ribosomes initiating at the AUG codon at the beginning of the GFP-encoding gene (141 nucleotides downstream of the M3 AUG codon in the expression construct). By IF microscopy, we found that GFP was distributed diffusely throughout pEGFP-transfected cells (Fig. 8B). Although fusion of GFP to the carboxyl terminus of ␮NS reduced the efficiency of ␮NS inclusion formation, globular inclusions were detected in approximately 80% of cells expressing ␮NSGFP (Fig. 8B). The other ␮NS-GFP-positive cells displayed a diffuse distribution for ␮NS-GFP that was similar to that for GFP alone (data not shown). In cells transfected with pEGFPM3(1–41), ␮NS(1–41)-GFP was diffusely distributed, demonstrating that the first 41 amino acids of ␮NS(T1L) are not sufficient for inclusion formation (Fig. 8B). When GFP was coexpressed with ␮2(T1L), GFP remained diffusely distributed in cells and did not colocalize with ␮2(T1L) (Fig. 8C). When ␮NS-GFP was coexpressed with ␮2(T1L), on the other hand, ␮NS-GFP reorganized into filamentous structures that colocalized with ␮2(T1L) (Fig. 8C). These structures were similar to those found when ␮NS(T1L) and ␮2(T1L) were coexpressed (Fig. 4A), demonstrating that the fusion of GFP to the carboxyl terminus of ␮NS(T1L) did not alter ␮NS colocalization with ␮2(T1L). When ␮NS (1–41)-GFP was coexpressed with ␮2(T1L), ␮NS(1–41)-GFP also colocalized with ␮2(T1L) in filamentous structures and in concentrated areas of the

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FIG. 6. Colocalization of ␮NS(T1L) and ␮2 in cotransfected cells treated with nocodazole. CV-1 cells were cotransfected with 1.75 ␮g of pCI-M3(T1L) and 0.25 ␮g of pCI-M1(T1L) (upper four panels) or 0.25 ␮g of pCI-M1(T3DN) (lower four panels) per well. Cells were left untreated or treated with 10 ␮M nocodazole added at 6 h p.t. (nocodazole), fixed at 18 h p.t., and immunostained for ␮NS (left column) and ␮2 (right column) as described in the legend to Fig. 3A. Bars, 10 ␮m.

nucleus (Fig. 8C). These results provide strong evidence that amino acids 1 to 41 of ␮NS are sufficient to support colocalization with ␮2. DISCUSSION Formation of globular phase-dense structures by ␮NS. Reovirus-infected cells contain phase-dense inclusions that are believed to be the sites of virus replication and assembly and are therefore referred to as viral factories. Little is known about the viral and cellular factors that are needed to form these structures. We found that transfected cells expressing ␮NS(T1L) or ␮NS(T3D) contain ␮NS globular inclusions that appear very similar to the globular viral factories in T3DNinfected cells (Fig. 1A). This finding supports our hypothesis (see introductory paragraphs) that ␮NS plays a major role in

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the formation and structure of viral factories in infected cells. We recognize, however, that these similarities do not formally prove that the structures are functionally equivalent. The inclusions formed in transfected cells after the overexpression of ␮NS may be the result of aggregation that coincidently forms structures with an appearance similar to that of globular viral factories. Misfolded proteins can accumulate in inclusions called aggresomes (18). These phase-dense structures are often ubiquitinated, surrounded by a collapsed vimentin intermediate filament network, and located in the perinuclear region (18). They require MTs to form but not to be maintained (18). The structures formed by ␮NS resemble aggresomes because they are phase dense (Fig. 1A), accumulate in the perinuclear region (Fig. 1A), and appear to use MTs to travel to this region; once formed, however, they are not disrupted by MT depolymerization (Fig. 2). We do not believe, however, that the ␮NS in globular inclusions is misfolded, because it can be relocalized upon ␮2(T1L) coexpression (Fig. 3A and 4A). Also, ␮NS inclusions are neither polyubiquitinated nor surrounded by a collapsed vimentin filament network (supplemental data can be found at http://micro.med.harvard.edu /nibert/suppl/broering02a/fig2.html). Future experiments, such as negative-stain EM of thin sections from M3-transfected and T3DN-infected cells, will allow further comparison of ␮NS globular inclusions and viral factories. The suggestion that viral nonstructural proteins form the structure of viral factories has been made for the Reoviridae family members rotavirus and bluetongue virus. IF microscopy was used to identify spherical inclusions that formed when rotavirus nonstructural proteins NSP5 and NSP2 were coexpressed in MA104 cells after transfection but not when the proteins were expressed individually (11). Negative-stain EM identified inclusions, similar in morphology to those in bluetongue virus-infected cells, in insect cells infected with a recombinant baculovirus that expresses bluetongue virus nonstructural protein NS2 (38). Rotavirus NSP5, rotavirus NSP2, and bluetongue virus NS2 can all be phosphorylated (10, 17, 36, 40) and are all proposed to hydrolyze nucleoside triphosphates (NTPs) (3, 16, 29, 36, 37). The phosphorylation status and NTP-hydrolyzing activities of ␮NS are uncharacterized. However, the M1 genome segment that encodes ␮2 was previously shown to determine differences in the NTPase activities of reovirus cores, and the ␮2 sequence includes regions with some similarity to the A and B motifs of NTPases (27). Investigating the similarities among these proteins may provide a better understanding of their capacities to form inclusions in the absence of other viral proteins. ␮NS and ␮2 association. The reovirus ␮2 protein was recently shown to play a role in determining the morphology of filamentous viral factories by associating with and stabilizing MTs, but ␮2 expression after transfection did not produce structures resembling viral factories (28). Similarly, when ␮NS was expressed in transfected cells, we found only globular inclusions (Fig. 1A). Filamentous structures resembling those in T1L-infected cells formed only upon coexpression of ␮2(T1L) and ␮NS (Fig. 4A). These results suggest that ␮NS and ␮2 cooperate in T1L-infected cells to determine the distribution and morphology of viral factories. The colocalization and redistribution of ␮NS and ␮2 in cotransfected cells strongly suggest an interaction between these proteins in vivo,

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FIG. 7. Distribution of ␮NS(41–721) in transfected cells with and without ␮2 coexpression. (A) CV-1 cells were transfected with 1 ␮g of pCI-M3(T1L) or pCI-M3(41–721) and 1 ␮g of pCI-neo as a carrier plasmid per well. CV-1 cells were infected (inf) with T1L at a multiplicity of infection of 5. Lysates were collected at 18 h p.t. or p.i. and analyzed by SDS-PAGE and immunoblotting with polyclonal antisera specific for full-length ␮NS (␣-␮NS) and the N-terminal 41 amino acids of ␮NS (␣-1–41 ␮NS). A band appearing in untransfected CV-1 cells is indicated on the right with an asterisk. The position of full-length ␮NS is indicated on the right. (B) Phase-contrast microscopy (left) and IF microscopy (right) of CV-1 cells transfected with 2 ␮g of pCI-M3(41–721) per well. Cells were fixed at 18 h p.t. and immunostained with Texas red-conjugated anti-␮NS rabbit IgG. (C) CV-1 cells were cotransfected with 1 ␮g of pCI-M3(41–721) and 1 ␮g of pCI-M1(T1L) (upper panels) or 1 ␮g of pCI-M3(41–721) and 1 ␮g of pCI-M1(T3DN) (lower panels) per well and fixed at 18 h p.t. Cells were immunostained for ␮NS (red) and ␮2 (green) as described in the legend to Fig. 3A. Bars, 10 ␮m.

either direct binding of the two proteins or indirect interaction through a cellular intermediate. The association of ␮NS and ␮2 is not dependent on the localization of ␮2 to MTs, because ␮NS and ␮2(T3DN) colocalized in transfected cells (Fig. 3A and 4A). The ␮NS/␮2 association is also not dependent on the capacity of ␮NS to form inclusions, since ␮NS(1–41)-GFP colocalized with ␮2(T1L) but did not form inclusions (Fig. 8). Only the N-terminal 41 amino acids of ␮NS(T1L) are needed to mediate the association with ␮2(T1L). Further evidence for an association between ␮NS and ␮2 was the increase in ␮2 expression in transfected cells when ␮NS(T1L) was coexpressed (Fig. 4B). This increased expression of ␮2 could be due to increased translation or increased stability of this protein, and studies to address these possibilities are under way. Previous genetic data also linked the M1 and M3 segments: in strains that accumulated deletions upon high passage, the capacity to accumulate deletions in M1 was mapped to M3 (6). In both this study and a previous one (28), ␮2 staining in the nucleus of M1-transfected cells was observed. The size of the

␮2 protein (83 kDa) exceeds the 60-kDa limit for passive diffusion into the nucleus (30). However, ␮2 contains predicted nuclear import and export signals (J. S. L. Parker, J. Kim, and M. L. Nibert, unpublished data) that may explain its distribution in the nucleus and the cytoplasm of transfected cells. Significant ␮2 staining in the nucleus of infected cells has not been reported (28). In this study, we found that coexpression of ␮NS reduced ␮2 staining in the nucleus of M3- and M1cotransfected cells (Fig. 3). As ␮NS (80 kDa) does not localize to the nucleus and does not have a known nuclear import signal, a reasonable explanation is that ␮NS sequesters ␮2 within cytoplasmic inclusions, thus reducing the amount of cytoplasmic ␮2 that is free to enter the nucleus. ␮NS(1–41)GFP, on the other hand, colocalized with ␮2 in the nucleus of cotransfected cells. As the ␮NS(1–41)-GFP fusion protein does not form inclusions (Fig. 8) and is smaller than the size limit for nuclear entry by passive diffusion, we hypothesize that ␮NS(1–41)-GFP enters the nucleus passively and is then retained there through its association with ␮2. These results

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suggest that ␮2 may enter the nucleus of infected cells, but further studies are needed to confirm that prediction and to assess any functional role that ␮2 nuclear localization may play in reovirus replication. In reoviruses with filamentous viral factories (22 of 24 strains tested in the study of Parker et al. [28]), the ␮NS/␮2 association may specifically function to recruit ␮NS to MTs and may be the first of multiple associations that bring together both viral and cellular factors to form viral factories. An association also occurs between ␮NS and ␮2(T3DN), which does not associate with MTs, suggesting that there may be other functions of the ␮NS/␮2 association. For example, ␮NS association with ␮2 could regulate the proposed NTPase (27) and RNA-binding (4) activities of ␮2. Similarly, activities of ␮NS such as core binding (5) or proposed RNA binding (1) could be affected by ␮NS association with ␮2. Much work remains to be done to determine the functions of ␮NS and ␮2 and how they alter each other’s activities in infected cells. Possible ␮NSC function. We found that ␮NS(41–721) forms inclusions but does not colocalize with ␮2 (Fig. 7), whereas ␮NS(1–41)-GFP does not form inclusions but colocalizes with ␮2(T1L) (Fig. 8). These observations identify a small region of ␮NS(T1L) (amino acids 1 to 41) that is necessary and sufficient (in terms of ␮NS regions) for ␮2 colocalization and a much larger region of ␮NS(T1L) (amino acids 41 to 721) that is necessary and sufficient (in terms of ␮NS regions) for inclusion formation. We believe that the ␮NS(41–721) protein is properly folded because it retains the capacity to form globular inclusions in transfected cells (Fig. 7B) and the capacity to bind to cores in vitro (T. J. Broering, P. L. Joyce, and M. L. Nibert, unpublished data). The results obtained with ␮NS(41–721) may be relevant to reovirus infection because the ␮NSC protein present in reovirus-infected L cells (20) is missing sequences from the N terminus of ␮NS and is postulated to comprise amino acids 41 to 721 of ␮NS (23, 41). Immunoblot analysis of reovirus-infected L cells with the N-terminal antiserum generated against amino acids 1 to 41 of ␮NS confirmed the absence of N-terminal amino acids in ␮NSC (T. J. Broering and M. L. Nibert, unpublished data). However, the analysis of ␮NSC in reovirus-infected CV-1 cells and recombinant baculovirus-infected insect cells has been complicated by the pres-

FIG. 8. Distribution of GFP, ␮NS-GFP, and ␮NS(1–41)-GFP in transfected cells with and without the coexpression of ␮2(T1L). (A) CV-1 cells were transfected with 1 ␮g of pEGFP, pEGFP-M3, or pEGFP-M3(1–41) and 1 ␮g of pCI-neo as a carrier plasmid per well. Lysates were collected at 18 h p.i. and analyzed by SDS-PAGE and immunoblotting with polyclonal anti-␮NS serum (␣-␮NS) or monoclonal anti-GFP IgG (␣-GFP). A band appearing in untransfected CV-1 cells is indicated on the left with an asterisk. The positions of ␮NS-GFP, ␮NS(1–41)-GFP, and GFP are indicated on the right. (B) CV-1 cells were transfected with 2 ␮g of pEGFP (upper left panel), pEGFP-M3 (upper right panel), or pEGFP-M3(1–41) (lower left panel), fixed at 18 h p.t., and immunostained with mouse monoclonal antibody to GFP followed by goat anti-mouse IgG conjugated to Alexa 488. (C) CV-1 cells were cotransfected with 0.25 ␮g of pEGFP (upper panels), pEGFP-M3 (middle panels), or pEGFP-M3(1–41) (lower panels) and 1.75 ␮g of pCI-M1(T1L) per well. Cells were fixed at 18 h p.t. and stained for GFP (left column) as described for panel B and ␮2 (right column) with rabbit anti-␮2 serum followed by goat anti-rabbit IgG conjugated to Alexa 594. Bars, 10 ␮m.

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FIG. 9. Model for viral factory formation. (A) When expressed in the absence of other viral proteins, ␮NS forms globular phase-dense inclusions that travel along MTs toward the nucleus. When ␮2(T3DN) is coexpressed with ␮NS, it is recruited to ␮NS globular inclusions. (B) In a T3DN-infected cell, ␮NS binds to cores and incorporates them into inclusions to generate a globular viral factory (VF). (C) ␮NS coats MTs when coexpressed with ␮2(T1L), forming filamentous structures. (D) In a T1L-infected cell, cores bound by ␮NS are recruited to MTs by the ␮NS/␮2 association, forming a filamentous VF.

ence of additional protein bands, between the ␮NS and ␮NSC bands, which react with the N-terminal ␮NS antiserum (Fig. 7A) (Broering and Nibert, unpublished). The compositions of these additional ␮NS bands are under investigation. If ␮NSC does indeed lack as much as 5 kDa of sequence from the N terminus of ␮NS, our data suggest a difference in ␮NS and ␮NSC activities in reovirus-infected cells. For example, if ␮NSC does not associate with ␮2(T1L), as the results obtained with ␮NS(41–721) suggest, it may alter the amount of inclusion material associated with MTs and may also be free to interact with other components. The relative levels of expression of ␮NS and ␮NSC may be regulated during infection to coordinate their different activities. Potential role for ␮NS in the formation of viral factories in infected cells: a current model. When expressed by transfection, ␮NS forms globular inclusions that can recruit coexpressed ␮2(T3DN) (Fig. 9A) (see Fig. 1A and 3A for data). These initially small inclusions may travel along MTs and coalesce to form large perinuclear inclusions (Fig. 9A) (see Fig. 1B and 2 for data). In cotransfected cells, ␮NS is recruited to MTs by an association with ␮2(T1L) (see Fig. 3A and 5 for data). We hypothesize that these two proteins form the coat

J. VIROL.

around MTs previously identified in infected cells (8) and that ␮2 mediates the previously identified association of ␮NS with the cytoskeletal fractions of infected cells (25) (Fig. 9C). Based on the capacity of ␮NS to bind to cores in vitro (5), the observation that cores are embedded within factories in infected cells (12, 31), and the isolation of core-like particles with ␮NS from infected cells (26), we also hypothesize that ␮NS may retain cores in viral factories as well as recruit unassembled core proteins. A viral factory may begin as a single transcribing core bound by ␮NS and grow as more ␮NS is added and new cores are assembled (Fig. 9B and D). We propose that the morphology and location of the viral factories are controlled through the ␮NS association with ␮2, which determines whether the factory is globular or filamentous (28) (Fig. 9). Based on our findings, we hypothesize that ␮NS initiates the formation and provides the structure for viral factories in infected cells. Reovirus particles, proteins, and RNA are then recruited to the sites of replication and particle assembly. By functioning as a scaffold, ␮NS may increase the local concentrations of reovirus proteins and RNA, organize the double-stranded RNA synthesis or particle assembly process, recruit specific cellular factors to contribute to these processes, and/or exclude other cellular factors from the area of reovirus assembly. The unique distributions of ␮NS and ␮2(T1L) when expressed individually and together are useful tools for identifying in vivo associations with other reovirus proteins and will allow us to test our hypothesis that ␮NS recruits other reovirus proteins and perhaps cellular proteins as well to inclusions. ACKNOWLEDGMENTS Many thanks are due to Laura Breun and Elaine Freimont for technical assistance, Aimee McCutcheon for assistance with plasmid construction, and Caroline Piggott for antibody titration. We are grateful to Darren Higgins and Angelika Gru ¨ndling for essential maintenance and advice regarding microscope facilities. This work was supported by NIH grants R29 AI-39533 and R01 AI-47904 (to M.L.N.) and by a USDA Hatch grant through the University of Wisconsin Extension (to M.L.N.). T.J.B. acknowledges previous support from predoctoral fellowships from the Wisconsin Alumni Research Foundation and NIH research training grant T32 GM0712 to the Molecular Biosciences Program at the University of Wisconsin—Madison. J.S.L.P. is the recipient of individual NRSA fellowship F32 AI-10134. REFERENCES 1. Antczak, J. B., and W. K. Joklik. 1992. Reovirus genome segment assortment into progeny genomes studied by the use of monoclonal antibodies directed against reovirus proteins. Virology 187:760–776. 2. Becker, M. M., M. I. Goral, P. R. Hazelton, G. S. Baer, S. E. Rodgers, E. G. Brown, K. M. Coombs, and T. S. Dermody. 2001. Reovirus ␴NS protein is required for nucleation of viral assembly complexes and formation of viral inclusions. J. Virol. 75:1459–1475. 3. Blackhall, J., A. Fuentes, K. Hansen, and G. Magnusson. 1997. Serine protein kinase activity associated with rotavirus phosphoprotein NSP5. J. Virol. 71:138–144. 4. Brentano, L., D. L. Noah, E. G. Brown, and B. Sherry. 1998. The reovirus protein ␮2, encoded by the M1 gene, is an RNA-binding protein. J. Virol. 72:8354–8357. 5. Broering, T. J., A. M. McCutcheon, V. E. Centonze, and M. L. Nibert. 2000. Reovirus nonstructural protein ␮NS binds to core particles but does not inhibit their transcription and capping activities. J. Virol. 74:5516–5524. 6. Brown, E. G., M. L. Nibert, and B. N. Fields. 1983. The L2 gene of reovirus serotype 3 controls the capacity to interfere, accumulate deletions and establish persistent infection, p. 275–288. In R. W. Compans and D. H. L. Bishop (ed.), Double-stranded RNA viruses. Elsevier Science Publishing Co., Inc., New York, N.Y. 7. Cashdollar, L. W., R. Chmelo, J. Esparza, G. R. Hudson, and W. K. Joklik. 1984. Molecular cloning of the complete genome of reovirus serotype 3. Virology 133:191–196.

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