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Journal of Virology, May 2005, p. 6194-6206, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6194-6206.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Carboxyl-Proximal Regions of Reovirus Nonstructural Protein µNS Necessary and Sufficient for Forming Factory-Like Inclusions

Teresa J. Broering ,1,2,{dagger},{ddagger} Michelle M. Arnold,1,3,{dagger} Cathy L. Miller,1 Jessica A. Hurt,4 Patricia L. Joyce,2,§ and Max L. Nibert1,3,4*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706,2 Ph.D. Programs in Virology,3 Biological and Biomedical Sciences, Division of Medical Sciences, Harvard University, Cambridge, Massachusetts 021384

Received 1 September 2004/ Accepted 14 January 2005


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ABSTRACT
 
Mammalian orthoreoviruses are believed to replicate in distinctive, cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. The viral nonstructural protein µNS has been implicated in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly. In this study, we sought to identify the regions of µNS that are involved in forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. Sequences in the carboxyl-terminal one-third of the 721-residue µNS protein were linked to this activity. Deletion of as few as eight residues from the carboxyl terminus of µNS resulted in loss of inclusion formation, suggesting that some portion of these residues is required for the phenotype. A region spanning residues 471 to 721 of µNS was the smallest one shown to be sufficient for forming factory-like inclusions. The region from positions 471 to 721 (471-721 region) includes both of two previously predicted coiled-coil segments in µNS, suggesting that one or both of these segments may also be required for inclusion formation. Deletion of the more amino-terminal one of the two predicted coiled-coil segments from the 471-721 region resulted in loss of the phenotype, although replacement of this segment with Aequorea victoria green fluorescent protein, which is known to weakly dimerize, largely restored inclusion formation. Sequences between the two predicted coiled-coil segments were also required for forming factory-like inclusions, and mutation of either one His residue (His570) or one Cys residue (Cys572) within these sequences disrupted the phenotype. The His and Cys residues are part of a small consensus motif that is conserved across µNS homologs from avian orthoreoviruses and aquareoviruses, suggesting this motif may have a common function in these related viruses. The inclusion-forming 471-721 region of µNS was shown to provide a useful platform for the presentation of peptides for studies of protein-protein association through colocalization to factory-like inclusions in transfected cells.


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INTRODUCTION
 
Viruses with ten-segmented, double-stranded RNA genomes from the family Reoviridae, genus Orthoreovirus, are believed to replicate in distinctive, cytoplasmic inclusion bodies (7, 10, 11, 13, 23, 24, 30, 33, 37-39, 41, 44, 45). These inclusions are commonly called viral factories (13, 30) or viroplasms (45) and are similar to cytoplasmic inclusions formed by other viruses in the same family. In cells infected by rotaviruses or orbiviruses, for example, these structures are called viroplasms (12, 40) or viral inclusion bodies (8, 43), respectively.

Many viruses sequester their replication machinery within localized structures or surfaces in infected cells: for example, herpes simplex virus (double-stranded DNA genome, family Herpesviridae) in nuclear inclusions (reviewed in reference 35), vaccinia virus (double-stranded DNA genome, family Poxviridae) in cytoplasmic inclusions (also called viral factories [reviewed in reference 29]), brome mosaic virus (single-stranded RNA [ssRNA] genome, family Bromoviridae) on the cytoplasmic face of the endoplasmic reticulum (32, 36), and flock house virus (ssRNA genome, family Nodaviridae) on the cytoplasmic face of mitochondria (27). The basis and consequences of such specific localizations are the subjects of active investigations in many laboratories. We anticipate that studies of mammalian orthoreovirus (reovirus) factories in our own laboratory will provide new insights into the still poorly characterized mechanisms for RNA packaging and replication by these and other viruses in the family Reoviridae, as well as on the general significance and mechanism of concentrating viral replication at particular sites within cells.

In early studies, reovirus factories were determined to contain fully and partially assembled viral particles, viral proteins, double-stranded RNA, microtubules, and "kinky" filaments proposed to be intermediate filaments but not membrane-bound structures or ribosomes (10, 11, 23, 33, 37, 39, 41). The factories have a peculiarly dense consistency that distinguishes them from the adjacent cytoplasm and causes them to appear highly refractile by phase-contrast microscopy. At least part of this property appears to reflect a protein matrix that suffuses the factories. The determinants or features of one or more viral proteins that would make it capable of forming such a matrix are not well understood but might involve a variety of different types of intersubunit interactions, as well as interactions with cellular factors.

Results from our laboratory and others have recently shown that a single reovirus protein, µNS, is sufficient for forming phase-dense globular inclusions in the cytoplasm of transfected cells (4, 7). The inclusions formed by µNS in such experiments are notably similar in appearance to globular reovirus factories formed in infected cells, as visualized by either phase-contrast or immunofluorescence (IF) microscopy (4, 7, 30), suggesting that µNS forms the matrix of the factories (7). Moreover, µNS can associate with several other reovirus proteins and recruit them to these inclusions in transfected cells. To date, the viral proteins that have been reported to be recruited to inclusions formed by reovirus µNS are microtubule-binding core protein µ2 (7); nonstructural and ssRNA-binding protein {sigma}NS (4, 26); and the core surface proteins {lambda}1, {lambda}2, and {sigma}2 (6). Whole core particles released into the cytoplasm during cell entry are also recruited to µNS inclusions (6). Indirect evidence suggests that µNS may possess ssRNA-binding activity as well, which may be important for recruiting the viral plus-strand RNAs to the factories and/or retaining them there for replication and packaging in infected cells (1). However, the ssRNA-binding activity of {sigma}NS is more firmly established (15-17, 19, 34, 42) and may play the more important role in ssRNA recruitment to or retention within the factories (4, 6, 26). Recent studies on the µNS homolog from avian orthoreoviruses have reached conclusions similar to these regarding the roles of µNS in forming the factory matrix and recruiting other viral proteins to the factories (44, 45). We explicitly specify in the present study when we intend a statement to encompass results for avian orthoreovirus; otherwise, we use the abbreviated name reovirus to represent mammalian orthoreovirus only.

The 80-kDa µNS protein is not a component of mature virions but is expressed to high levels in infected cells and is concentrated in the reovirus factories (7, 49). Other previous findings about µNS include its association with transcriptionally active particles isolated from infected cells (28), its binding to the surfaces of purified core particles in vitro (5), and its capacity to recruit entering core particles to inclusions (6). The µNS protein has two predicted coiled-coil segments in the carboxyl-terminal one-third of its sequence (25), but the oligomeric status of µNS has not been demonstrated. Little else is known about the structure of µNS, although domains have been predicted from the pattern of conserved and variable regions of sequence among the µNS alleles from different reovirus isolates (25). An isoform of µNS lacking ~5 kDa from its amino terminus and called µNSC is also present in virus-infected cells (22, 46). The origin of µNSC is not certain, but it is proposed to result from either secondary initiation or cleavage near Met41 in µNS. A recombinant protein engineered to lack the first 40 residues of µNS, µNS(41-721), which should be similar to µNSC, also forms phase-dense globular inclusions in transfected cells (7).

Involvement of microtubules in forming reovirus factories has also been demonstrated. The M1 segment, which encodes the structurally minor core protein µ2, has been shown to determine both the timing of factory formation (24) and the morphology of factories (30). The filamentous morphology of factories formed by most reovirus strains has been attributed to microtubule association of the µ2 protein (30). When µNS and µ2 are coexpressed in cells without other viral proteins, µNS is redistributed with µ2 to filamentous inclusions (7), suggesting that these two proteins together determine the formation and morphology of filamentous viral factories. Aside from any effects of µ2, however, µNS also requires microtubules for undergoing condensation of its smaller inclusions into larger ones, as shown by the effects of the microtubule-depolymerizing drug nocodazole. This probably reflects a role for microtubule-based transport in the formation of µNS inclusions and the determination of viral factory morphology (7, 30).

By forming the matrix of viral factories, as well as recruiting other components to these structures, the reovirus µNS protein could serve to concentrate the components for viral replication and assembly, to arrange the components in specific ways to promote replication and assembly, and/or to sequester the components and their mediated functions from antiviral factors in the cellular environment. To better understand the role of µNS in forming the matrix of viral factories, we engineered constructs to express a panel of µNS truncations and point mutants and thereby determined the regions of this protein that are involved in forming factory-like inclusions in transfected cells. The results identified several different sequences in the C-terminal one-third of µNS that are necessary and sufficient for this activity of µNS.


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MATERIALS AND METHODS
 
Cells and antibodies. CV-1 cells were maintained in Dulbecco modified Eagle medium (Invitrogen Life Technologies) containing 10% fetal bovine serum (HyClone Laboratories) and 10 µg of gentamicin solution (Invitrogen Life Technologies) per ml. Goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG conjugated to Alexa 488 or Alexa 594 were obtained from Molecular Probes. Rabbit polyclonal antisera to µNS or µ2 have been described previously (5, 7, 30). For some experiments, we used protein A-purified rabbit anti-µNS or anti-µ2 IgG conjugated to Texas Red, Oregon Green, Alexa 488, or Alexa 594 by using kits obtained from Molecular Probes. Mouse monoclonal antibody (MAb) FK2 against conjugated ubiquitin (14) was purchased from Medical & Biological Laboratories. Mouse MAb JL8 against Aequorea victoria green fluorescent protein (GFP) was purchased from BD Biosciences. Mouse MAb HA.11 against an immunodominant epitope of influenza A virus hemagglutinin (HA) was purchased from Covance. Mouse MAb 3E10 specific for reovirus {sigma}NS protein (3) was a gift from T. S. Dermody and coworkers (Vanderbilt University). All antibodies were titrated to optimize signal-to-noise ratios.

Expression constructs. Reovirus proteins were expressed from genes cloned into the mammalian expression vector pCI-neo (Promega). pCI-M3(T1L) and pCI-M3(T3D) to express µNS from type 1 Lang (T1L) or type 3 Dearing (T3D) reovirus have been described previously (7), as have pCI-M3(41-721) to express µNS residues 41 to 721 (7), pCI-M1(T1L) to express µ2 (30), and pCI-S3(T1L) to express {sigma}NS (26). Vent polymerase, which was used for all PCRs, and other enzymes were from New England Biolabs unless otherwise stated.

To express µNS residues 1 to 683, site-directed mutagenesis was used to introduce a stop codon at nucleotides 2068 to 2070, followed by a Bsu36I site. QuikChange site-directed mutagenesis (Stratagene) was used according to the manufacturer's protocol with pFastBac-M3(T1L) (5) as a template, forward primer 5'-GGATACGATGAACTAACCTCAGGCTAAATCATTGCG-3', and reverse primer 5'-CGCAATGATTTAGCCTGAGGTTAGTTCATCGTATCC-3' (double underline, nucleotide change to add the stop codon; single underline, nucleotide change to add the restriction site). The region containing the desired mutations was excised by digestion with HindIII and then ligated to pFastBac-M3(T1L) that had been cut with HindIII to remove the same region, generating pFastBac-M3(1-683). The subcloned region was sequenced to confirm its correctness. The M3 gene was removed from pFastBac-M3(1-683) by digestion with SalI and NheI and then ligated to pCI-neo that had been cut with the same enzymes, generating pCI-M3(1-683).

To generate other µNS truncations, start and stop codons and restriction sites were introduced at different positions in the M3 gene by PCR amplification of the desired M3 region. The truncations were made in either T1L or T3D M3. We have found no difference in inclusion formation with T1L and T3D µNS (7, 26), suggesting that the truncations from these allelic µNS proteins are directly comparable. In addition, the T1L and T3D µNS proteins are ~96% identical (25). Each PCR was performed with pGEM-4Z-M3(T1L) or pGEM-4Z-M3 (T3D) (5) as a template, and the primers are listed in Table 1. Each PCR product was cut with the restriction enzymes listed in Table 1 and then ligated to a plasmid that had been cut with these same enzymes, the plasmid being either pGEM-4Z (for pGEM-4Z-M3(1-173) and pGEM-4Z-M3(1-221) or the template plasmid (for all other constructs). The correctness of each construct was confirmed by sequencing. The truncated M3 genes were subcloned into pCI-neo by using the restriction enzymes listed in Table 2. Each construct was named for the residues of µNS that the expressed protein should ultimately contain (Tables 1 and 2).


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  		TABLE 1. Construction of plasmids with truncated M3 genes


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  		TABLE 2. Construction of plasmids to express µNS truncations

The pEGFP-N1 and pEGFP-C1 vectors (BD Biosciences) were respectively used to express fusions of enhanced A. victoria GFP to the C or N terminus of the desired µNS region. pEGFP-N1-M3(T1L) was previously constructed to express GFP fused to the C terminus of µNS (µNS/GFP) (7). To express GFP fused to the N terminus of µNS residues 471 to 721, pGEM-4Z-M3(471-721) was cut with SalI and KpnI, and the excised fragment was then ligated to pEGFP-C1 that had been cut with the same enzymes, generating pEGFP-C1-M3(471-721). To express GFP fused to the N termini of even smaller C-terminal regions of µNS, an EcoRI site was introduced at the desired position in the M3 gene during PCR amplification. Each PCR was performed with pGEM-4Z-M3(T1L) (5) as a template, the forward primers listed in Table 3, and a reverse primer complementary to the 3' end of the M3 coding strand with an added BamHI site (underlined) (5'-GCAGGGGATCCGATGAATGGGGGTCGGGAAGGCTTAAGGG-3'). Each PCR product was cut with EcoRI and BamHI and was then ligated to pEGFP-C1 that had been cut with the same enzymes. The correctness of each construct was confirmed by sequencing, and the construct was named for the residues of µNS that the expressed protein contains (Table 3).


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  		TABLE 3. Construction of plasmids to express µNS-GFP fusions

To generate HA-tagged fusions, epitope-encoding forward primer 5'-CGTAGCTAGCGTCATGGCTTACCCATACGACGTCCCAGACTACGCTCTCGAGATGC-3' and reverse primer 5'-GCATCTCGAGAGCGTAGTCTGGGACGTCGTATGGGTAAGCCATGACGCTAGCTACG-3' (underlining indicates NheI and XhoI sites added to each primer) were annealed by boiling in 1x SSC (150 mM NaCl plus 15 mM sodium citrate [pH 7.0]), followed by cooling at room temperature for 1 h. The duplex oligonucleotide and vector pCI-neo were then digested with NheI and XhoI, and the products were ligated to generate pCI-neo-HA. The region encoding µNS(561-721) was amplified by PCR from the template pCI-M3(T1L) by using forward primer 5'-GCTAGAATTCATGTAGTCTGGATATGTATTTGAGACACCAC-3' and reverse primer 5'-GATCGATCCCGGGTCGGGAAGGCTTAAGGGATTAGGGCAA-3' (underlining indicates an EcoRI or SmaI site added to each primer). The PCR product and vector pCI-neo-HA were then sequentially digested with SmaI and EcoRI, and the products were ligated to generate pCI-neo-HA-M3(561-721). The region encoding µNS(471-721) was amplified by PCR from the template pCI-M3(T1L) by using the forward primer 5'-AGCTCTCGAGGTCATGTCCAGTGACATGGTAGACGGGATTAAAC-3' (underlining indicates an added XhoI site) and reverse primer 5'-CGAAGCATTAACCCTCAC-3'. The PCR product and vector pCI-neo-HA were then digested with XhoI and NotI, and the products were ligated to generate pCI-neo-HA-M3(471-721). To generate untagged µNS(561-721), PCR was performed by using the template pCI-M3(T1L), forward primer5'-AGCTGAATTCGTCATGGCTTGTAGTCTGGTATGTATTTGAGACAC-3' (underline indicates an added EcoRI site), and reverse primer 5'-CGAAGCATTAACCCTCAC-3'. The PCR product and vector pCI-neo were then digested with EcoRI and NotI, and the products were ligated to generate pCI-neo-M3(561-721). The correctness of the final constructs was confirmed by sequencing.

To generate a µNS(471-721) or full-length µNS protein with residues His569 and His570 changed to glutamine and Cys572 changed to serine, QuikChange site-directed mutagenesis was used according to the manufacturer's protocol with pCI-M3(471-721) or pCI-M3(T3D) as a template, forward primer 5'-GGATATGTATCTGCGACAACAAACTTCCATTAATGGTCATGC-3', and reverse primer 5'-GCATGACCATTAATGGAAGTTTGTTGTCGCAGATACATATCC-3' (double underlines, nucleotide changes to provide amino acid changes; single underline, nucleotide change to remove a DdeI site for screening purposes). After the QuikChange protocol, the mutated pCI-M3(471-721) or pCI-M3(T3D) plasmid was prepared for subcloning by cutting with NheI and EcoRI or by cutting with BlpI and NotI, respectively. The excised fragments were then ligated to pCI-neo or pCI-M3(T3D), respectively, that had been cut with the same respective pair of enzymes, generating the final versions of pCI-M3(471-721)QQS and pCI-M3(T3D)QQS. The correctness of these constructs was confirmed by sequencing. To generate a full-length µNS protein with Cys561 or Cys572 changed to Ser or His569, His570, or His576 changed to Gln, QuikChange site-directed mutagenesis was used according to the manufacturer's protocol with pCI-M3(T3D) as a template and the primers listed in Table 4. According to the QuikChange protocol, each mutated plasmid was prepared for subcloning by cutting with BlpI and NotI. In each case, the excised fragment was then ligated to pCI-M3(T3D) that had been cut with the same enzymes, generating the constructs as named in Table 4. The correctness of each subcloned region was confirmed by sequencing.


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  		TABLE 4. Construction of plasmids to express µNS point mutants

To generate a fusion of µNS residues 1 to 41 to the N terminus of GFP and µNS residues 471 to 721 to the C terminus of GFP, DNA encoding residues 1 to 41 of µNS and a large portion of GFP was removed from pEGFP-N1-M3(1-41) (7) by digestion with NheI and BsrGI. pEGFP-C1-M3(471-721) was also cut with these enzymes, and the two DNA fragments were ligated to generate pEGFP-M3(1-41)/GFP/M3(471-721). To generate a fusion of µNS residues 1 to 12 to the N terminus of GFP and µNS residues 471 to 721 to the C terminus of GFP, forward primer 5'-AATTC ATGGCTTCATTCAAGGGATTCTCCGTCAACACTGTTGCG-3' and reverse primer 5'-GATCCGCAACAGTGTTGACGGAGAATCCCTTGAATGAAGCCATG-3' were annealed to generate a small duplex encoding µNS residues 1 to 12 and having BamHI and EcoRI overhangs at the respective ends (underlined). This small duplex and plasmid pEGFP-N1 were both digested with BamHI and EcoRI and then ligated to yield pEGFP-N1-M3(1-12). This plasmid was then digested with NheI and BsrGI, and the excised fragment was ligated to pEGFP-C1-M3(471-721) that had been digested with the same enzymes, generating plasmid pEGFP-M3(1-12)/GFP/M3(471-721). The correctness of the resulting construct was confirmed by sequencing.

Transfections and IF microscopy. Cells were seeded the day before transfection at a density of 1.5 x 104 per cm2 in six-well plates (9.6 cm2 per well) containing glass coverslips (19 mm). Cells were transfected with 2 µg of DNA and 7 µl of Lipofectamine 2000 (Invitrogen Life Technologies) or 1.5 µl of DNA and 10 µl of Polyfect (Qiagen) according to the manufacturer's directions. Cells were further incubated for 18 to 24 h at 37°C before fixation for 10 min at room temperature in 2% paraformaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4 [pH 7.5]) or 3 min at –20°C in ice-cold methanol. Fixed cells were washed three times with PBS, permeabilized, and blocked in PBS containing 1% bovine serum albumin and 0.1% Triton X-100 (PBSAT). Primary antibodies were diluted in PBSAT and incubated with cells for 25 to 40 min at room temperature. After three washes in PBS, secondary antibodies diluted in PBSAT were added and incubated with cells for 25 min at room temperature. Coverslips were incubated with 300 nM 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes) in PBS for 5 min to counterstain cell nuclei, briefly washed in PBS, and mounted on glass slides with Prolong (Molecular Probes). Samples were examined by using a TE-300 inverted microscope (Nikon) equipped with phase and fluorescence optics, and images were collected digitally as described elsewhere (30). All images were processed and prepared for presentation by using Photoshop (Adobe Systems).

Immunoblot analysis. CV-1 cells were transfected as described for IF, and whole-cell lysates were collected 18 to 24 h posttransfection (p.t.). CV-1 cells (1.2 x 106) were washed briefly in PBS and then scraped into 1 ml of PBS and pelleted. The pelleted cells were resuspended in 30 µl of PBS containing protease inhibitors (Roche Biomedicals), lysed into sample buffer, boiled for 10 min, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electroblotted from the gels to nitrocellulose in 25 mM Tris and 192 mM glycine (pH 8.3). Binding of antibodies was detected with alkaline phosphatase-coupled goat anti-mouse IgG (Bio-Rad Laboratories) and the colorimetric reagents p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Bio-Rad Laboratories).

Sequence comparisons. The following µNS and µNS-homolog sequences, each designated as complete coding sequences in GenBank, were compared: µNS of mammalian orthoreoviruses T1L (accession no. AAF13169), type 2 Jones (accession no. AAF13170), and T3D (accession no. AAF13171); µNS of avian orthoreoviruses 1733 (accession no. AAQ81873), S1133 (accession no. AAS78987), 1017-1 (accession no. AAS78988), 2408 (accession no. AAS78990), 601G (accession no. AAS78991), 750505 (accession no. AAS78992), 916SI (accession no. AAS78993), 918 (accession no. AAS78994), 919 (accession no. AAS78995), OS161 (accession no. AAS78996), R2 (accession no. AAS78997), and T6 (accession no. AAS78998); and NS1 of aquareoviruses grass carp 873 (accession no. AAM92735) and golden shiner (accession no. AAM92747). Sequences were compared by using the Bestfit and Pretty programs from the Genetics Computer Group Wisconsin package. The Multicoil program (http://multicoil.lcs.mit.edu/cgi-bin/multicoil) (48) was used for predicting coiled-coil regions.


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RESULTS
 
The C terminus of µNS is required for forming factory-like inclusions. We have previously shown that a truncated µNS protein lacking the N-terminal 40 residues, µNS(41-721), collects in cytoplasmic, factory-like inclusions in transfected cells, similarly to full-length µNS (7). The N terminus of µNS is thus not required for forming these structures. To determine whether the C terminus of µNS is required, we engineered M3 gene constructs to express a series of truncated µNS proteins that lacked increasing numbers of residues from the C terminus (Table 2). Each of these plasmids was transfected into CV-1 cells, and the cell lysates were subjected to SDS-PAGE, followed by immunoblotting with polyclonal anti-µNS serum (5). The results verified the expression of an appropriately sized, µNS-derived protein from each construct (data not shown).

To determine the intracellular distribution of the C-terminally truncated µNS proteins, CV-1 cells were separately transfected with each of the plasmids and later fixed and immunostained with polyclonal anti-µNS antibodies (Fig. 1A, left and right columns). In addition, the cells were coimmunostained with MAb FK2, which recognizes conjugated ubiquitin (14), to determine whether any of these proteins were substantially misfolded and targeted for degradation by the ubiquitin-proteasome system (Fig. 1A, center and right columns). Each of the C-terminally truncated proteins was diffusely distributed in the cytoplasm and nucleus, in clear contrast to full-length µNS, which collected in globular inclusions as expected (Fig. 1A and data not shown). We conclude that all of these truncated proteins are negative for forming factory-like inclusions (summarized in Fig. 2) and thus that some portion of the smallest region we deleted from the C terminus of µNS, residues 714 to 721, is required for inclusion formation. None of the truncated proteins appeared to be aggregated or strongly colocalized with conjugated ubiquitin (Fig. 1A and data not shown), suggesting that they were not substantially misfolded.



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  		FIG. 1. IF microscopy of C- terminally truncated µNS proteins. CV-1 cells were transfected with plasmids to express the indicated proteins. The cells were then fixed at 18 h p.t. for subsequent immunostaining. Nuclei were counterstained with DAPI (blue). Scale bars, 10 µm. (A) The cells were immunostained both with rabbit anti-µNS IgG conjugated to Texas red (left column, red in right column) and with mouse MAb FK2 for conjugated ubiquitin (cUb), followed by goat anti-mouse IgG conjugated to Alexa 488 (center column, green in right column). (B) The cells were immunostained both with rabbit anti-µNS IgG followed by goat anti-rabbit IgG conjugated to Alexa 594 (left column, red in right column) and with rabbit anti-µ2 IgG conjugated to Alexa 488 (center column, green in right column).



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  		FIG. 2. Summary of µNS truncations and their activities. The full-length µNS protein is indicated by a horizontally elongated black bar spanning residues 1 to 721 (positions numbered above and below). The µNS truncation mutants are also shown as black bars spanning the approximate portion of µNS that each represents. Enhanced A. victoria GFP fused to the N or C terminus of µNS in some cases is represented by an open bar. An influenza virus HA epitope fused to the N terminus of µNS in some cases is represented by an open circle. Approximate extents of the coiled-coil segments predicted by Multicoil are indicated by vertically elongated gray bars. The capacity of each protein to form factory-like inclusions in transfected cells (Inc) and to colocalize with T1L µ2 in transfected cells (µ2) is indicated as positive (+), negative (–), or not determined (nd). Localized structures that costained for conjugated ubiquitin were concluded to be aggregates of misfolded protein (agg). The results for µNS(1-721), µNS(1-721)/GFP, µNS(1-41)/GFP, µNS(14-721), and µNS(41-721) have been reported previously (7, 26).

All of the C-terminally truncated µNS proteins appeared to be partially localized to the nucleus (Fig. 1A, left column), even though µNS(1-683), µNS(1-700), and µNS(1-713) were above the 60-kDa limit for passive diffusion through nuclear pores (reviewed in reference 31) (Table 2). In addition, at least some of the truncated proteins appeared to be excluded from nucleoli (Fig. 1A, left column). The relevance of this nuclear pattern is unclear because full-length µNS is not detected in the nuclei of infected or M3-transfected cells (7, 30). When coexpressed with µ2(T1L), each of the C-terminally truncated proteins strongly colocalized with µ2 on microtubules (Fig. 1B and data not shown; summarized in Fig. 2). This was expected because each protein included µNS residues 1 to 41, which are known to be sufficient for association with µ2 (7, 26).

The N-terminal 470 residues of µNS are not required for forming factory-like inclusions but affect inclusion shape. To identify the minimal region of µNS required for inclusion formation, we engineered M3 gene constructs to express a series of truncated µNS proteins that lacked increasing numbers of residues from the N terminus (Table 2). Each of these plasmids was transfected into CV-1 cells, and the cell lysates were subjected to SDS-PAGE, followed by immunoblotting with the anti-µNS serum (5). The results verified expression of an appropriately sized, µNS-derived protein from each construct (data not shown).

The intracellular distribution of each of the N-terminally truncated µNS proteins was determined as described above for the C-terminal truncations, including coimmunostaining with MAb FK2. With the exception of µNS(363-721), which showed a distinctly aggregated pattern and strongly colocalized with conjugated ubiquitin (Fig. 3), each of the N-terminally truncated proteins collected in globular inclusions (Fig. 3, left and right columns, and data not shown). However, the proteins missing more than the 40 N-terminal residues of µNS often formed inclusions with more elongated shapes and enclosed fenestrations than those formed by full-length µNS or µNS(41-721) (Fig. 3, left and right columns). Although these inclusions had altered morphologies, they did not colocalize with conjugated ubiquitin (Fig. 3, center and right columns), suggesting that these truncated proteins were not substantially misfolded. The behavior of µNS(363-721), on the other hand, suggested that it was in fact largely misfolded.



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  		FIG. 3. IF microscopy of N-terminally truncated µNS proteins. CV-1 cells were transfected with plasmids to express the indicated proteins. The cells were then fixed and stained as described for Fig. 1A. Scale bars, 10 µm.

When coexpressed with µ2(T1L), none of the N-terminally truncated µNS proteins colocalized with µ2 on microtubules or in inclusions (data not shown; summarized in Fig. 2). This was expected because each of these proteins lacks µNS residues 14 to 40, which are known to be required for association with µ2 (7, 26). From the results summarized in Fig. 2, we conclude that µNS residues 471 to 721 are a sufficient part of µNS for forming factory-like inclusions. These residues encompass the two predicted coiled-coil segments of µNS, the intervening "linker" between these segment, and the C-terminal "tail" that follows the second predicted coiled-coil segment (25). Given the altered morphologies of the inclusions formed by µNS proteins lacking more than the 40 N-terminal residues, we also conclude that some portion of residues 41 to 172 plays a distinguishable role in modulating inclusion morphology.

Residues 561 to 721 of µNS are sufficient for allowing a GFP-tagged protein, but not an HA-tagged or untagged protein, to form factory-like inclusions. To determine whether a region of µNS smaller than residues 471 to 721 may be sufficient for inclusion formation, and also to allow more ready detection of these smaller proteins, we next generated constructs to express GFP fused to the N terminus of selected µNS truncations (Table 3). Each of these plasmids was transfected into CV-1 cells, and the cell lysates were subjected to SDS-PAGE, followed by immunoblotting with GFP-specific MAb JL-8. The results verified production of an appropriately sized, GFP- and µNS-derived fusion protein from each construct (Fig. 4A).



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  		FIG. 4. Immunoblotting and IF microscopy of GFP-tagged derivatives of µNS. CV-1 cells were transfected with plasmids to express GFP or µNS-GFP fusions as indicated and then analyzed at 18 h p.t. by immunoblotting (A) or IF microscopy (B). Scale bars, 10 µm. (A) Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with mouse MAb JL8 for GFP followed by goat anti-mouse IgG conjugated to alkaline phosphatase. Positions of molecular weight markers are indicated (in kilodaltons) to the left of each panel. (B) After fixation, cells were immunostained with mouse MAb JL8 for GFP, followed by goat anti-mouse IgG conjugated to Alexa 488.

To determine the intracellular distribution of each fusion protein, CV-1 cells were transfected and later immunostained with the GFP-specific MAb. Nonfused GFP was diffusely distributed in the cytoplasm and nucleus, and GFP fused to the C terminus of full-length µNS (µNS/GFP) collected in globular inclusions as previously shown (7) (also see Fig. 4B). GFP fused to the N terminus of µNS(471-721) [GFP/µNS(471-721)] also collected in inclusions (Fig. 4B), which appeared similar to those formed by µNS(471-721) (Fig. 3) or µNS/GFP (Fig. 4B). GFP fused to the N terminus of µNS(561-721) collected in inclusions as well, although a small fraction of the transfected cells (~13%) displayed a diffuse distribution of this protein (Fig. 4B and data not shown). In contrast, GFP fused to the N terminus of µNS(614-721), µNS(625-721), or µNS(695-721) was diffusely distributed in the cytoplasm and nucleus of most or all cells expressing them (Fig. 4B and data not shown). From these results, we conclude that residues 561 to 721 are a sufficient part of µNS in GFP fusions for forming factory-like inclusions (summarized in Fig. 2), albeit at a lower efficiency than full-length µNS or µNS(471-721). The first predicted coiled-coil segment (25) is thus dispensable for inclusion formation by a µNS-GFP fusion. On the other hand, some portion of residues 561 to 613, in the linker between the two predicted coiled-coil segments, is required.

Given that A. victoria GFP is known to weakly dimerize (9) and also that GFP/µNS(561-721) was less efficient at forming factory-like inclusions than was GFP/µNS(471-721), we hypothesized that the GFP tag may partially complement an otherwise-required contribution of the first predicted coiled-coil segment of µNS for forming inclusions. We therefore tested another version of µNS(561-721), this one fused to an epitope of influenza virus HA (47) at its N terminus [HA/µNS(561-721)]. After expression in transfected CV-1 cells and immunostaining with tag-specific MAb HA.11, HA/µNS(561-721) was diffusely distributed in the cytoplasm and nucleus and thus not concentrated in inclusions (Fig. 5). Fusion of the HA tag to the N terminus of µNS(471-721) [HA/µNS(471-721)], in contrast, caused little or no reduction in its capacity to form factory-like inclusions (Fig. 5). We also generated and tested a third version of µNS(561-721), this one lacking any tag. After expression in transfected CV-1 cells and immunostaining with anti-µNS antibodies, untagged µNS(561-721) was diffusely distributed in the cytoplasm and nucleus and thus not concentrated in inclusions (Fig. 5). Untagged µNS(471-721) tested in parallel formed factory-like inclusions (Fig. 5) as seen in the preceding experiments (see Fig. 3). Based on these results, we conclude that the more N-terminal predicted coiled-coil segment of µNS is required for inclusion formation in the absence of a fusion tag such as GFP that can independently self-associate.



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  		FIG. 5. IF microscopy of HA-tagged or untagged versions of µNS(561-721) and µNS(471-721). CV-1 cells were transfected with plasmids to express the proteins as indicated. The cells were then fixed at 18 h p.t. and immunostained either with mouse MAb HA.11 for the influenza virus HA epitope, followed by goat anti-mouse IgG conjugated to Alexa 488 (left panels), or with rabbit anti-µNS IgG, followed by goat anti-rabbit IgG conjugated to Alexa 594 (right panels). Scale bars, 10 µm.

Putative metal-chelating residues His570 and Cys572 are required for factory-like inclusion formation. In an effort to identify specific residues in µNS that may be required for inclusion formation, we compared the deduced protein sequences of µNS homologs from mammalian and avian orthoreoviruses, as well as from aquareoviruses (see Materials and Methods for the GenBank accession numbers) (2, 25, 45). In all, the µNS homologs derived from 17 isolates in the three groups: 3 mammalian isolates from the Orthoreovirus genus, 12 avian isolates from the Orthoreovirus genus, and 2 piscine isolates from the Aquareovirus genus. The overall identity scores for any two µNS homologs from separate groups are small: <30% in each case (2, 45) (the present study and data not shown). Despite this degree of divergence, the C-terminal one-third of each of these µNS homologs contains two predicted coiled-coil segments, of similar lengths and spacing, separated by a linker and followed by a C-terminal tail (data not shown), as previously described for the mammalian and avian isolates (25, 45).

Interestingly, in the linker between the predicted coiled-coil segments, we identified a small consensus motif common to all of the examined µNS homologs (Fig. 6A). This sequence,Ile/Leu-x-x-Tyr-Leu-x-x-His-Thr/Val-Cys-Ile/Val-Asn (where"x" represents nonconserved positions), includes two residues (underlined) with strong potential to chelate transition metal ions such as Zn2+. The two residues correspond to His570 and Cys572 in the mammalian orthoreovirus µNS proteins. Each of the µNS homologs contains other His and/or Cys residues flanking the consensus motif, but the position and spacing of these residues is not conserved among the examined sequences (Fig. 6A). In mammalian orthoreovirus µNS, the other conserved His and Cys residues in this region are Cys561, His569, and His576. The consensus motif spans residues 563 to 574 in the mammalian orthoreovirus µNS proteins and is thus near the beginning of the minimal C-terminal region of µNS that we showed to be sufficient for inclusion formation in GFP fusions (Fig. 4).



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  		FIG. 6. Sequence conservation in the "linker" region of µNS homologs and IF microscopy of µNS proteins with mutations at His and/or Cys residues in the consensus motif. (A) Sequence conservation. Sequences are shown in single-letter code, with position numbers indicated at left and right. The consensus motif was defined by comparing the illustrated region from the µNS homologs of 3 mammalian orthoreoviruses (mORV), 12 avian orthoreoviruses (aORV) (all identical in this region), and 2 aquareoviruses (AqRV) (both identical in this region). Conserved positions are highlighted by being boxed. Asterisks indicate conserved positions corresponding to His570 and Cys572 in mammalian orthoreovirus µNS. These and other His and Cys residues in or near the consensus motif are highlighted by being circled. (B) IF microscopy. CV-1 cells were transfected with plasmids to express the indicated proteins. The cells were then fixed at 18 h p.t. and immunostained both with rabbit anti-µNS IgG conjugated to Oregon Green and with mouse MAb FK2 for conjugated ubiquitin, followed by goat anti-mouse IgG conjugated to Alexa 594. Nuclei were counterstained with DAPI. Only the anti-µNS staining is shown since no colocalization with FK2 staining was apparent. Scale bars, 10 µm.

To determine whether the conserved residues with metal-chelating potential in the consensus motif are important for inclusion formation, we first generated constructs encoding Gln (Q) substitutions for both His570 and the adjacent residue, His569, as well as a Ser (S) substitution for Cys572 (Fig. 6A). The constructs were generated with all three of these mutations in the setting of either full-length µNS or µNS(471-721). We then transfected CV-1 cells with these mutant plasmids and costained the cells with anti-µNS antibodies and MAb FK2 for conjugated ubiquitin. Both proteins, designated µNS(1-721)QQS and µNS(471-721)QQS, were diffusely distributed in the cytoplasm and nucleus (Fig. 6B and data not shown), and neither strongly colocalized with conjugated ubiquitin (data not shown). This distribution was in sharp contrast to the inclusions in which both full-length µNS and µNS(471-721) concentrated (data not shown for this experiment, but see previous figures), demonstrating that one or more of residues His569, His570, and Cys572 is important for inclusion formation.

We next generated constructs encoding single mutations at residues Cys561 (to Ser), His569 (to Gln), His570 (to Gln), Cys572 (to Ser), or His576 (to Gln) within full-length µNS. The mutant plasmids were transfected into CV-1 cells, and the cells were costained with anti-µNS antibodies and MAb FK2 for conjugated ubiquitin. Both µNS(1-721)H570Q and µNS(1-721)C572S were diffusely distributed in the cytoplasm and nucleus (Fig. 6B). In contrast, µNS(1-721)C561S, µNS(1-721)H569Q, and µNS(1-721)H576Q all collected in globular inclusions indistinguishable from those formed by wild-type µNS (Fig. 6B). None of the mutant proteins strongly colocalized with conjugated ubiquitin (data not shown). From these findings, we conclude that His570 and Cys572 are specifically required for µNS to form factory-like inclusions in transfected cells.

{sigma}NS recruitment to factory-like inclusions containing µNS residues 1 to 12 connected by GFP to µNS residues 471 to 721. We have previously shown that {sigma}NS colocalizes with full-length µNS but not with µNS(41-721) or µNS(13-721) in cotransfected cells, indicating that the N terminus of µNS is required for recruiting {sigma}NS (7, 26). Because both {sigma}NS and the N-terminal 41 residues of µNS fused to the N terminus of GFP [µNS(1-41)/GFP] are diffusely distributed in cells, we encountered difficulties in using IF microscopy to determine whether the N terminus of µNS is sufficient for association with {sigma}NS (7, 26). Upon finding in the present study that GFP/µNS(471-721) collected in factory-like inclusions in transfected cells (Fig. 4B), we recognized that this protein provided a new platform on which to assay protein-protein associations through redistribution to its distinctive inclusions. When GFP/µNS(471-721) was coexpressed with {sigma}NS, {sigma}NS remained diffusely distributed in the cytoplasm and nucleus and did not colocalize with the GFP/µNS(471-721) inclusions (Fig. 7A). This was expected, because some portion of µNS residues 1 to 13 is required for recruiting {sigma}NS to µNS inclusions (26).



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  		FIG. 7. IF microscopy of {sigma}NS coexpressed with µNS-GFP fusions. (A) CV-1 cells were transfected with plasmids to express the proteins indicated. The cells were then fixed at 18 h p.t. and immunostained with {sigma}NS-specific mouse MAb 3E10, followed by goat anti-mouse IgG conjugated to Alexa 594 (center column, red in right column). GFP-containing fusions were visualized directly (left column, green in right column). Nuclei were counterstained with DAPI (blue). Scale bars, 10 µm. (B) Summary of the fusions and their activities. See Fig. 2 legend for explanations of most details. Dashed lines indicate internally deleted regions of µNS. The capacity of each protein to colocalize with T1L {sigma}NS in transfected cells ({sigma}NS) is indicated as positive (+), negative (–), or unknown (?). The results for µNS(1-721), µNS(1-721)/GFP, µNS(1-41)/GFP, µNS(14-721), and µNS(41-721) have been reported previously (7, 26).

To determine whether µNS residues 1 to 41 can direct recruitment of {sigma}NS to the inclusions of GFP/µNS(471-721), we constructed a plasmid to express these residues fused to the N terminus of GFP/µNS(471-721). When expressed in transfected CV-1 cells, this protein, µNS(1-41)/GFP/µNS(471-721), collected in inclusions similar to those formed by µNS(471-721) or GFP/µNS(471-721) (data not shown). When {sigma}NS was coexpressed with µNS(1-41)/GFP/µNS(471-721) and coimmunostained with anti-{sigma}NS MAb 3E10 (3) and the anti-µNS serum, {sigma}NS strongly colocalized with the µNS(1-41)/GFP/µNS(471-721) inclusions (Fig. 7A). This was in stark contrast to the diffuse distribution of {sigma}NS expressed alone (26) or coexpressed with GFP/µNS(471-721) (Fig. 7A). When coexpressed with µ2, µNS(1-41)/GFP/µNS(471-721) was redistributed to filamentous inclusions (data not shown), as expected because of the presence of µNS residues 14 to 40 (26) (also see Fig. 2 and 7B). These results newly demonstrate that µNS residues 41 to 470 are dispensable for recruiting {sigma}NS (summarized in Fig. 7B).

We additionally constructed a plasmid to express µNS residues 1 to 12 fused to the N terminus of GFP/µNS(471-721). When expressed in transfected CV-1 cells, this protein, µNS (1-12)/GFP/µNS(471-721), collected in factory-like inclusions (data not shown). Moreover, when coexpressed, {sigma}NS was strongly recruited to the µNS(1-12)/GFP/µNS(471-721) inclusions (Fig. 7A). When coexpressed with µ2, µNS(1-12)/GFP/µNS(471-721) was not redistributed to filamentous inclusions (data not shown), as expected because of the absence of µNS residues 13 to 41 (26). These results demonstrate that µNS residues 13 to 470 are dispensable for recruiting {sigma}NS (summarized in Fig. 7B) and suggest that µNS residues 1 to 12 may be sufficient for this association.


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DISCUSSION
 
Numerous observations suggest that the inclusion bodies in reovirus-infected cells are sites of viral replication and assembly (4, 6, 33, 38, 39). The roles of nonstructural protein µNS in forming the matrix of these viral factories and recruiting other components to them have become a focus of recent studies in this field (4, 6, 7). Given the recent findings and the absence of strong evidence for other functions of this protein, we hypothesize that the primary role of µNS in reovirus infection is to build and organize the factories to promote replication and assembly. We find it intriguing that reovirus may have a protein dedicated to this role, and we therefore wish to learn more about how the different regions of µNS carry out specific activities toward this end.

Current summary of µNS functional regions. Truncation and deletion analyses have been a fruitful approach for identifying discrete regions of µNS involved in its different activities. For example, the current study demonstrated that the C-terminal one-third of µNS (residues 471 to 721) is a sufficient part of this protein for forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. The present study also identified several smaller regions of sequence, and even single residues, within this third of µNS that are required for inclusion formation.

An otherwise-uncharacterized region of µNS (residues 41 to 172) affected the shape of the inclusions in the present study, causing them to be more compact and less fenestrated when present in the inclusion-forming protein. This effect could reflect a direct interaction of some portion of residues 41 to 172 with the C-terminal, inclusion-forming region of µNS or with some cellular protein, which in an unknown manner produces more compact inclusions. This effect could also reflect an indirect mechanism, such as a reduction in the turnover rate of the inclusion-forming protein such that holes do not develop within the inclusions.

Previous reports have shown that the N-terminal 40 residues of µNS are involved in associations with at least two other reovirus proteins: microtubule-binding protein µ2 and ssRNA-binding protein {sigma}NS (7, 26). Moreover, the specific residues necessary or sufficient for these activities have been shown to be separable. Residues 1 to 41 of µNS are sufficient for association with µ2 (7), but only some portion of residues 14 to 40 of µNS is necessary for this activity (26). In contrast, some portion of residues 1 to 13 of µNS is necessary for association with {sigma}NS (26), and residues 1 to 12 of µNS may be sufficient for this activity (the present study). Other, complementary evidence suggests the N-terminal 40 residues of µNS represent a discrete domain. A form of µNS lacking ~5 kDa of N-terminal sequence is produced in infected cells concomitantly with the full-length protein (7, 46). Although the origin of this smaller form, called µNSC, remains in question, the fact that it is similar to µNS(41-721), which lacks both µ2 and {sigma}NS association activities, suggests that it and full-length µNS may play distinguishable roles in infection. Interestingly, a µNSC-like form of µNS has been recently identified in avian orthoreovirus and found not to associate with the avian orthoreovirus {sigma}NS protein as well, suggesting that the different activities of µNS and µNSC in this regard may be a conserved point of regulation for reoviruses in general (45). The avian orthoreovirus µ2 protein has not yet been examined for µNS association.

Except for the modulation of inclusion shape described in the present study and a predicted coiled-coil segment from residues 518 to 561 (25), the large central portion of µNS spanning residues 41 to 560 remains without well-characterized activities. This region from positions 41 to 560 may contain the residues responsible for µNS associations with other reovirus components, including the individual core-surface proteins, {lambda}1, {lambda}2, and {sigma}2 and whole core particles (6, 7). In fact, these activities are shared by µNS and µNSC, indicating they do not require the N-terminal 40 residues of µNS. Further truncation/deletion analyses are needed to determine whether each of the individual core-surface proteins, as well as core particles, may associate with µNS/µNSC through a distinct set of residues, such as those for µ2 and {sigma}NS in the unique N-terminal region of µNS.

Other components with which µNS/µNSC may yet be shown to associate include the reovirus RNA-dependent RNA polymerase {lambda}3, the reovirus outer-capsid proteins, the reovirus RNA molecules, and any possible number of cellular factors. For each of these potential associations, again, further truncation/deletion analyses could aid in ascertaining whether each component may associate with µNS or µNSC through a distinct set of residues in µNS. In any case, the emerging picture of µNS is one of a protein involved in multiple interactions to form the factory matrix and to recruit other components to the factories, with a modular organization of its primary sequence for performing these many distinct activities.

How does µNS form factory-like inclusions? There are many possible interactions that could be involved in forming the three-dimensional structure that each inclusion likely represents, even before the addition of other viral components as found in the factories in infected cells. For example, µNS-µNS interactions may be all that are required for forming factory-like inclusions. Alternatively, interactions of µNS with one or more cellular factor may be necessary, with the cellular factor(s) acting as bridges between µNS monomers or oligomers. The presence of predicted coiled-coil segments in µNS has led to the suggestion that µNS forms a basal small oligomer, possibly a dimer, through {alpha}-helical coiled-coil interactions (25). If so, then inclusions are likely built by linking these small oligomers together, with or without the help of cellular factors. In any case, according to the new results, µNS(471-721) must be capable of mediating or instigating all of the necessary interactions for inclusion formation. The smaller regions within µNS(471-721) that are required for forming inclusions might be directly involved in these interactions.

The consensus motif that spans residues 563 to 574 of µNS and that is partially conserved in the homologous proteins of avian orthoreoviruses and aquareoviruses includes residues His570 and Cys572, which are required for inclusion formation. Given that His and Cys residues have strong potential to chelate transition metal ions such as Zn2+, we hypothesize that metal chelation by His570 and Cys572 is a part of their role in forming inclusions. Moreover, considering that His570 and Cys572 are so closely spaced and that nearby residues with the same potential for metal chelation—Cys561, His569, and His576—are dispensable for inclusion formation, we hypothesize that His570 and Cys572 form half of an intermolecular metal-chelating motif, similar to the zinc hook of Rad50 (18) or the zinc clasp of CD4/8 and Lck (20, 21). The latter two motifs provide four zinc-chelating residues, two from each participating subunit, and contribute to homodimerization in the case of Rad50 or heterodimerization in the case of CD4/8 and Lck. Of course, further work is needed to determine whether µNS residues His570 and Cys572 indeed chelate a metal ion and, if so, whether this chelation may contribute to µNS homodimerization or µNS heterodimerization with a cellular protein, either of which could be required for inclusion formation.

Some portion of the C-terminal eight residues of µNS (Phe-Ser-Val-Pro-Thr-Asp-Glu-Leu) is also required for inclusion formation. Since this region contains no His or Cys residues, it must contribute to forming inclusions by a mechanism distinct from that hypothesized for His570 and Cys572. Although this region is not highly conserved in the homologous proteins of avian orthoreoviruses and aquareoviruses, each of the µNS homologs terminates in Leu and has at least one acidic residue and no basic residues in its C-terminal eight positions. Further work is needed to determine whether these C-terminal residues may contribute to µNS-µNS interaction or to µNS interaction with a cellular protein, either of which could be additionally required for inclusion formation.

Evidence in the present study suggests that the more N-terminal of the predicted coiled-coil segments in µNS (25) is required for inclusion formation but that the loss of function resulting from its deletion can be largely rescued by fusion to GFP. We interpret this result to indicate that the role of the first predicted coiled-coil segment is in self-association, which GFP can also mediate (9), but we acknowledge that this interpretation requires further testing. Although the more C-terminal one of the two predicted coiled-coil segments in µNS has not been directly tested for its role in inclusion formation, we expect that at least part of it is also required. Coiled-coil interactions between µNS subunits could, for example, mediate the formation of basal oligomers, which then interact through other motifs to form the inclusions. Alternatively, one or both of the coiled-coil segments could mediate hetero-oligomerization with a cellular protein. As noted in Results, predicted coiled-coil segments are also found flanking the proposed metal chelation sequences in each of the µNS homologs from avian orthoreoviruses and aquareoviruses, suggesting a conserved function for these motifs.

Formation of µNS inclusions as a tool to study protein-protein associations inside cells. The characteristic subcellular localization of µNS and its inclusion-forming derivatives—in cytoplasmic, phase-dense, globular inclusions—has proven a potent tool for studying associations between µNS and other viral proteins in infected or transfected cells (4, 6, 7). In the present study, we acquired original evidence that GFP/µNS(471-721) may provide a useful platform for examining possible associations between any two proteins in transfected cells. In particular, full-length proteins or protein regions expressed as fusions to GFP/µNS(471-721) should localize to the distinctive, globular inclusions induced by the region from positions 471 to 721. The capacity of this fused protein or protein region to associate with another, "test" protein can then be assayed by examining cells in which the test protein has been coexpressed with the inclusion-forming fusion protein. If the test protein localizes to the globular inclusions, then it and the fused protein or protein region can be concluded to associate. Of course, nonfused GFP/µNS(471-721) should be tested in parallel as a negative control for the specificity of test protein localization to the inclusions. A range of relative expression levels of the two proteins could also be tested, in case relative overexpression of the test protein may retard inclusion formation by the other. We are currently extending our studies of the feasibility of this approach as a general one for studying protein-protein associations within the "native" cellular environment in which the proteins normally reside and function.


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ACKNOWLEDGMENTS
 
We express our sincere gratitude to Elaine Freimont and Jason Dinoso for laboratory maintenance and technical assistance and to other members of our lab for helpful discussions. We also thank John Patton and John Parker for reviews of a draft of the manuscript.

This study was supported in part by NIH grants R01 AI47904 (M.L.N.) and F32 AI56939 (C.L.M.) and by a junior faculty grant from the Giovanni Armenise-Harvard Foundation (M.L.N.). T.J.B. and M.M.A. were also supported by NIH grant T32 AI07245 to the Viral Infectivity Training Program. J.A.H. was also supported by NIH grant T32 GM07226 to the Biological and Biomedical Sciences Training Program. At earlier stages of this work, C.L.M. was also supported by NIH grant T32 AI07061 to the Combined Infectious Diseases Training Program.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department 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. Back

{dagger} T.J.B. and M.M.A. contributed equally to this study. Back

{ddagger} Present address: Massachusetts Biologic Laboratories, Jamaica Plain, MA 02130. Back

§ Present address: Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Back


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Journal of Virology, May 2005, p. 6194-6206, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6194-6206.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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