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Previous Article | Next Article 
Journal of Virology, August 2002, p. 8285-8297, Vol. 76, No. 16
0022-538X/02/$04.00+0 DOI: 10.1128/JVI.76.16.8285-8297.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Jonghwa Kim,1,2 and Max L. Nibert1*
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin 537062
Received 28 February 2002/ Accepted 9 May 2002
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ABSTRACT |
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INTRODUCTION |
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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 µ2, 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 µ2 and NS (2). It was suggested that viral RNA-protein complexes containing NS nucleate inclusion formation and that other viral proteins, including µ2, 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 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 W28-7, 2001; C. L. Miller, T. J. Broering, J. S. L. Parker, and M. L. Nibert, unpublished data).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 µ2-encoding M1 genome segment (28). When µ2 derived from T1L [µ2(T1L)] is expressed in cells, it colocalizes with MTs and causes MT bundling and stabilization, whereas µ2 derived from T3DN [µ2(T3DN)] does not (28). These findings implicate protein µ2 as a major determinant of viral factory association with MTs. However, µ2 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, µNS, 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 µNS and core complex resembled the morphology of factories in thin sections of reovirus-infected cells viewed by EM (33). By immunofluorescence (IF) microscopy, µNS was found concentrated within viral factories in infected cells (25, 28). Based on this evidence, we hypothesized as part of the current study that µNS plays a major role in viral factory structure and morphogenesis in infected cells.
µNS is an 80-kDa protein expressed at high levels in infected cells (41, 42). A second form of the protein, called µNSC, is also produced (20). Chemical cleavage of µNS and µNSC from infected cells demonstrated that µNSC lacks about 5 kDa of sequence from the amino (N) terminus of µNS (41). It was hypothesized that the synthesis of µNS results from ribosome initiation at the first AUG codon in the M3 plus-strand RNA (nucleotides 19 to 21) whereas the synthesis of µNSC 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 µNSC formation in infected cells indicate that µNS and µNSC do not have a precursor-product relationship (41), supporting the hypothesis that µNSC arises by secondary initiation rather than by cleavage of full-length µNS. The relative functions of µNS and µNSC in infected cells are not known. To address the hypothesis that µNS and µNSC 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 µNS and µ2 when coexpressed suggest that specific associations between these two proteins may be key to forming viral factories and recruiting the factors necessary for replication and assembly.
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MATERIALS AND METHODS |
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Antibodies. Monoclonal antibodies to Cy3-conjugated ß-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 µNS (5) and µ2 (described below). µNS and µ2 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 µNS antiserum did not stain cells expressing µ2 or GFP, and the GFP antibodies did not stain cells expressing µNS or µ2. The µ2 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 µNS or GFP in transfected cells.
Rabbit polyclonal antiserum specific for µ2 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). µ2 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-ß-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 µ2. To further purify µ2 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 µNS was produced by using a fusion protein of glutathione S-transferase (GST) and amino acids 1 to 41 of the T1L µNS protein [GST-µNS(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-µNS(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 µNS 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).
Generation of a µNS(41-721) expression construct. To generate a vector to direct the expression of a µNS 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 µNS 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 µNS and an additional 4 amino acids. We have not been able to detect this product in immunoblots with µNS antisera (data not shown). Ribosome initiation at the second AUG codon should produce a protein containing amino acids 41 to 721 of µNS, with a predicted molecular mass of 76 kDa. We have detected this product (see Fig. 7).
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To generate a full-length µNS 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 x 104 per cm2 in six-well plates (9.6 cm2 per well) containing glass coverslips. Transfections were performed with 2 µg of DNA (total) and 6 µl of Lipofectamine (Life Technologies) per well according to the manufacturer's directions. Infections were done for 1 h at room temperature with 200 µl 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 µNS 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 x 106) were washed briefly in PBS, scraped into 2 ml of PBS, and pelleted. The pelleted cells were resuspended in 60 µl 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-4-chloro-3-indolylphosphate p-toluidine salt (Bio-Rad).
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RESULTS |
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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 µNS 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 µNS 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 µNS inclusions are not aggresomes.
Size and distribution of µNS(T1L) globular inclusions are dependent on MTs. Based on the previous observations that MTs are embedded in reovirus factories (8, 12, 35) and that µNS may associate with MTs (25), we examined samples for the colocalization of µNS(T1L) with MTs by staining pCI-M3(T1L)-transfected cells for µNS and ß-tubulin. µNS 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).
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µNS and µ2 colocalize when coexpressed in cells. The µNS globular inclusions in transfected cells expressing µNS(T1L) contrast with the filamentous distribution of µNS in T1L-infected cells (28) (Fig. 1A, compare top and bottom panels). Because the filamentous appearance of viral factories in T1L-infected cells is determined by the capacity of µ2(T1L) to associate with MTs (28), we hypothesized that µ2 associates with µNS and redistributes it to MTs. To test this hypothesis, we transfected CV-1 cells with pCI-M3(T1L) and pCI-M1(T1L) at a 1:1 (wt/wt) ratio and examined the distribution of µNS and µ2 by using protein-specific polyclonal antisera. The µNS(T1L) and µ2(T1L) proteins colocalized in thick filamentous structures that surrounded the nucleus (Fig. 3A). The distribution of µNS(T1L) and µ2(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 µNS(T1L) and µ2(T1L) associate and that µ2(T1L) likely mediates the redistribution of µNS(T1L) to MTs.
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As described previously, some of the µ2 protein—especially µ2(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 µ2(T1L) or µ2(T3DN) in cells coexpressing µNS(T1L), we found that the nuclear association of µ2 was much less evident (Fig. 3A). This result suggests the existence of an equilibrium between µ2 proteins in different subcellular compartments (nucleus and cytoplasm) that is altered by the association of µ2 with µNS.
Varying the relative levels of µNS and µ2 expression alters the morphology of µNS/µ2 structures. Although the globular inclusions containing µNS(T1L) and µ2(T3DN) closely resemble the globular factories in T3DN-infected cells, the filamentous structures containing µNS(T1L) and µ2(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 µNS/µ2 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, µNS(T1L) colocalized with µ2 (Fig. 4A ); however, the relative amounts of transfected DNA affected the morphology of µNS/µ2 structures. When more of the µNS expression plasmid was transfected relative to the µ2 expression plasmid (8:1), globular inclusions connected by thin filaments were predominantly seen with µNS(T1L) and µ2(T1L) and large globular perinuclear inclusions were predominantly seen with µNS(T1L) and µ2(T3DN) (Fig. 4A). Alternatively, when less of the µNS expression plasmid was transfected relative to the µ2 expression plasmid (1:8), the morphology of µNS/µ2 structures was closer to that of µ2 alone: distributed filaments for µ2(T1L) and small perinuclear structures, with rough edges, for µ2(T3DN) (Fig. 4A). In addition, the association of µ2 with the nucleus was more evident in these cells (Fig. 4A). Structures containing µNS and µ2 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.
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µNS(T1L) is colinear with MTs in the presence of µ2(T1L) but not in the presence of µ2(T3DN). It was previously shown that µ2(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 µNS/µ2 inclusions with MTs by costaining transfected cells for µNS and ß-tubulin (Fig. 5). Similar to previous findings for T1L-infected cells (28), we did not find significant colocalization of µNS(T1L) with MTs, but we did find that the finer filamentous structures in cells coexpressing µNS(T1L) and µ2(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 tubulin-specific antibody (supplemental data can be found at http://micro.med.harvard.edu/nibert/suppl/broering02a/fig3.html), suggesting that the µNS(T1L)/µ2(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 µNS and ß-tubulin (Fig. 5). µNS 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 µNS(T1L) expressed in the absence of other viral proteins (Fig. 2). We obtained similar results when cells were cotransfected with µNS and µ2 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 µNS(T1L)/µ2(T1L) inclusions associate with MTs.
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The µNS N terminus is required for colocalization with µ2. A second form of µNS, called µNSC, is found in reovirus-infected cells and is missing amino acids from the N terminus of µNS (41). To determine whether the N terminus of µNS is required for inclusion formation and colocalization with µ2, 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 µNS(T1L) [µNS(41-721)]. The protein expressed in cells upon transfection of pCI-M3(41-721) reacted with the full-length µNS polyclonal antiserum but was not recognized by a polyclonal antiserum generated to the first 41 amino acids of µNS (Fig. 7A); these results confirm that the N terminus of µNS is absent from µNS(41-721). Full-length µNS expressed in transfected cells and in reovirus-infected cells was recognized by both full-length and N-terminal µNS polyclonal antisera (Fig. 7A). Phase-contrast microscopy of CV-1 cells expressing µNS(41-721) revealed globular phase-dense structures that contained µNS(41-721), as determined by IF microscopy (Fig. 7B). Therefore, amino acids 1 to 40 of µNS are not required for the formation of globular inclusions in transfected cells.
When µNS(41-721) was coexpressed with µ2(T1L), the two proteins did not colocalize in filamentous structures (Fig. 7C). Rather, when coexpressed with µ2(T1L), µNS(41-721) was seen in globular inclusions similar to those seen when the protein was expressed without µ2 (Fig. 7B), and µ2(T1L) had a filamentous and nuclear distribution similar to that seen when it was expressed without µNS (28) (Fig. 3B). µNS(41-721) and µ2(T3DN) also failed to colocalize (Fig. 7C). Smooth-edged globular structures containing µNS(41-721), similar to those found without µ2 coexpression (Fig. 7B), were surrounded by µ2(T3DN)-containing rough-edged structures; this appearance was similar to the pattern of µ2(T3DN) expressed in the absence of other reovirus proteins (28) (Fig. 3B). We conclude that amino acids 1 to 40 of µNS(T1L) are required for colocalization with µ2 but not for inclusion formation.
µNS(T1L) amino acids 1 to 41 are sufficient for µ2(T1L) colocalization. To determine whether the N-terminal amino acids of µNS that are required for µ2 colocalization are sufficient for colocalization, we constructed a plasmid [pEGFP-M3(1-41)] that expresses amino acids 1 to 41 of µNS(T1L) fused to the N terminus of GFP [µNS(1-41)-GFP]. To control for any effect of GFP on µNS/µ2 colocalization or inclusion formation, a plasmid (pEGFP-M3) was constructed that expresses full-length µNS(T1L) fused to the N terminus of GFP (µNS-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 µNS 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 µNS recognized µNS(1-41)-GFP and µNS-GFP but not GFP (data not shown). By immunoblotting, a protein of about the same size as native GFP was detected with the µNS(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, µNS(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).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µNS are uncharacterized. However, the M1 genome segment that encodes µ2 was previously shown to determine differences in the NTPase activities of reovirus cores, and the µ2 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.
µNS and µ2 association. The reovirus µ2 protein was recently shown to play a role in determining the morphology of filamentous viral factories by associating with and stabilizing MTs, but µ2 expression after transfection did not produce structures resembling viral factories (28). Similarly, when µNS 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 µ2(T1L) and µNS (Fig. 4A). These results suggest that µNS and µ2 cooperate in T1L-infected cells to determine the distribution and morphology of viral factories. The colocalization and redistribution of µNS and µ2 in cotransfected cells strongly suggest an interaction between these proteins in vivo, either direct binding of the two proteins or indirect interaction through a cellular intermediate. The association of µNS and µ2 is not dependent on the localization of µ2 to MTs, because µNS and µ2(T3DN) colocalized in transfected cells (Fig. 3A and 4A). The µNS/µ2 association is also not dependent on the capacity of µNS to form inclusions, since µNS(1-41)-GFP colocalized with µ2(T1L) but did not form inclusions (Fig. 8). Only the N-terminal 41 amino acids of µNS(T1L) are needed to mediate the association with µ2(T1L). Further evidence for an association between µNS and µ2 was the increase in µ2 expression in transfected cells when µNS(T1L) was coexpressed (Fig. 4B). This increased expression of µ2 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), µ2 staining in the nucleus of M1-transfected cells was observed. The size of the µ2 protein (83 kDa) exceeds the 60-kDa limit for passive diffusion into the nucleus (30). However, µ2 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 µ2 staining in the nucleus of infected cells has not been reported (28). In this study, we found that coexpression of µNS reduced µ2 staining in the nucleus of M3- and M1-cotransfected cells (Fig. 3). As µNS (80 kDa) does not localize to the nucleus and does not have a known nuclear import signal, a reasonable explanation is that µNS sequesters µ2 within cytoplasmic inclusions, thus reducing the amount of cytoplasmic µ2 that is free to enter the nucleus. µNS(1-41)-GFP, on the other hand, colocalized with µ2 in the nucleus of cotransfected cells. As the µNS(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 µNS(1-41)-GFP enters the nucleus passively and is then retained there through its association with µ2. These results suggest that µ2 may enter the nucleus of infected cells, but further studies are needed to confirm that prediction and to assess any functional role that µ2 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 µNS/µ2 association may specifically function to recruit µNS 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 µNS and µ2(T3DN), which does not associate with MTs, suggesting that there may be other functions of the µNS/µ2 association. For example, µNS association with µ2 could regulate the proposed NTPase (27) and RNA-binding (4) activities of µ2. Similarly, activities of µNS such as core binding (5) or proposed RNA binding (1) could be affected by µNS association with µ2. Much work remains to be done to determine the functions of µNS and µ2 and how they alter each other's activities in infected cells.
Possible µNSC function. We found that µNS(41-721) forms inclusions but does not colocalize with µ2 (Fig. 7), whereas µNS(1-41)-GFP does not form inclusions but colocalizes with µ2(T1L) (Fig. 8). These observations identify a small region of µNS(T1L) (amino acids 1 to 41) that is necessary and sufficient (in terms of µNS regions) for µ2 colocalization and a much larger region of µNS(T1L) (amino acids 41 to 721) that is necessary and sufficient (in terms of µNS regions) for inclusion formation. We believe that the µNS(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 µNS(41-721) may be relevant to reovirus infection because the µNSC protein present in reovirus-infected L cells (20) is missing sequences from the N terminus of µNS and is postulated to comprise amino acids 41 to 721 of µNS (23, 41). Immunoblot analysis of reovirus-infected L cells with the N-terminal antiserum generated against amino acids 1 to 41 of µNS confirmed the absence of N-terminal amino acids in µNSC (T. J. Broering and M. L. Nibert, unpublished data). However, the analysis of µNSC in reovirus-infected CV-1 cells and recombinant baculovirus-infected insect cells has been complicated by the presence of additional protein bands, between the µNS and µNSC bands, which react with the N-terminal µNS antiserum (Fig. 7A) (Broering and Nibert, unpublished). The compositions of these additional µNS bands are under investigation. If µNSC does indeed lack as much as 5 kDa of sequence from the N terminus of µNS, our data suggest a difference in µNS and µNSC activities in reovirus-infected cells. For example, if µNSC does not associate with µ2(T1L), as the results obtained with µNS(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 µNS and µNSC may be regulated during infection to coordinate their different activities.
Potential role for µNS in the formation of viral factories in infected cells: a current model. When expressed by transfection, µNS forms globular inclusions that can recruit coexpressed µ2(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, µNS is recruited to MTs by an association with µ2(T1L) (see Fig. 3A and 5 for data). We hypothesize that these two proteins form the coat around MTs previously identified in infected cells (8) and that µ2 mediates the previously identified association of µNS with the cytoskeletal fractions of infected cells (25) (Fig. 9C). Based on the capacity of µNS 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 µNS from infected cells (26), we also hypothesize that µNS 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 µNS and grow as more µNS 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 µNS association with µ2, which determines whether the factory is globular or filamentous (28) (Fig. 9). Based on our findings, we hypothesize that µNS 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, µNS 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 µNS and µ2(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 µNS recruits other reovirus proteins and perhaps cellular proteins as well to inclusions.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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Present address: Department of Radiation Oncology, University of North Carolina—Chapel Hill, Chapel Hill, NC 27599.
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REFERENCES |
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NS protein is required for nucleation of viral assembly complexes and formation of viral inclusions. J. Virol. 75:1459-1475.
-µNS) and the N-terminal 41 amino acids of µNS (
