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Journal of Virology, February 2004, p. 1882-1892, Vol. 78, No. 4
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.4.1882-1892.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Reovirus Nonstructural Protein µNS Recruits Viral Core Surface Proteins and Entering Core Particles to Factory-Like Inclusions

Teresa J. Broering,1 Jonghwa Kim,1,2 Cathy L. Miller,1 Caroline D. S. Piggott,1,3 Jason B. Dinoso,1 Max L. Nibert,1* and John S. L. Parker1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin 53706,2 Harvard College Research Program, Harvard University, Cambridge, Massachusetts 021383

Received 30 July 2003/ Accepted 28 October 2003


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ABSTRACT
 
Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein µNS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with µNS. We found that all three of these proteins—{lambda}1, {lambda}2, and {sigma}2—localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with µNS, on the other hand, each core surface protein colocalized with µNS in globular inclusions, supporting the initial hypothesis. We also found that {lambda}1, {lambda}2, and {sigma}2 each localized to filamentous inclusions formed upon the coexpression of µNS and µ2, a structurally minor core protein that associates with microtubules. The first 40 residues of µNS, which are required for association with µ2 and the RNA-binding nonstructural protein {sigma}NS, were not required for association with any of the three core surface proteins. When coexpressed with µ2 in the absence of µNS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with µ2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of µNS that had been preformed in those cells, providing evidence that µNS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins µNS and µ2 in this process.


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INTRODUCTION
 
The molecular machinery used for viral replication in the cytoplasm or nucleus of infected cells is commonly concentrated and organized in distinct sites or structures (reviewed in references 22 and 27). Schwartz et al. (44) recently proposed that all viruses that replicate through mRNA intermediates, including double-stranded RNA (dsRNA) viruses, may sequester their mRNA templates within a multiprotein complex that either is attached to cellular membranes or forms a distinct core-like structure. By doing so, these viruses may concentrate the minus-strand RNA products for use as templates, while limiting the exposure of dsRNA intermediates or products to host cell defense mechanisms, such as protein kinase R, RNase L, and the factors that mediate RNA interference (13, 43, 44).

The nonfusogenic mammalian orthoreoviruses (reoviruses) sequester their segmented dsRNA genomes, together with the viral polymerase molecules and capping enzymes for mRNA synthesis, within a 52-MDa core particle (38, 41). This core displays T=1 icosahedral symmetry and is composed of the following five viral proteins: {lambda}1 and {sigma}2, which form the core shell and decorating nodules (41); {lambda}2, the mRNA capping guanylyltransferase and methyltransferase, which forms a turret on the exterior of the core shell around each fivefold axis (12, 32, 41, 54); and {lambda}3 and µ2, the RNA-dependent RNA polymerase (14, 51, 52) and its cofactor (57), respectively, which are situated internal to the shell near the fivefold axes (15). The 10 dsRNA genome segments are also packaged inside the shell, where they can be used as templates for mRNA synthesis by the viral transcriptases (2, 5, 46). Over the course of reovirus infection, many new core particles are assembled and presumably then coated with the three remaining viral outer capsid proteins to produce infectious progeny virions (36, 47). In addition, some or all of the newly assembled cores synthesize more of the viral mRNAs, thereby amplifying the production of viral genes, gene products, and particles (23, 25, 29).

How the core is assembled remains poorly understood. It is a seemingly complex process that involves multiple events as follows (relative timing is not implied by the listed order): (i) formation of an icosahedral protein shell from {lambda}1 and {sigma}2, (ii) addition of the {lambda}2 mRNA capping enzyme turret outside this shell, (iii) addition of the {lambda}3 polymerase and µ2 cofactor inside the shell, (iv) assortment and packaging of the 10 distinct mRNA molecules, and (v) one round of minus-strand synthesis from each of the mRNA templates to regenerate the 10 dsRNA genome segments (reviewed in references 39 and 58). Packaging and minus-strand synthesis may be linked (1, 59), and the manner by which the internal components are placed inside the shell or by which the shell forms around the internal components remains a mystery. Despite these uncertainties, the assembly of cores and the replication of viral RNA are believed to occur within distinct structures that form in the cytoplasm of reovirus-infected cells and are commonly referred to as viral inclusions or factories (3, 4, 8, 33, 35, 40, 42, 45, 48, 49).

We and others have recently identified several determinants of reovirus factory formation and morphology. Nearly all reovirus strains examined to date form microtubule-associated filamentous factories that are similar to those of the type 1 prototype strain, Lang (T1L) (40). In contrast, the type 3 prototype strain, Dearing, in use in our lab (T3DN) forms globular factories that lack filaments (40). Analysis of a panel of T1L/T3D reassortants identified the M1 genome segment, which encodes the structurally minor core protein µ2, as the genetic determinant of this difference in factory morphology (40). The µ2 protein from reovirus strains that form filamentous factories was identified as a microtubule-associated protein, which determines this morphology (40). Another recent study identified µ2 as the primary determinant of a difference in the rates of formation of viral factories in T1L- versus T3D-infected cells (33). We have further found that when the reovirus nonstructural protein µNS is expressed in cells in the absence of other viral proteins, it forms large globular inclusions that are indistinguishable by phase-contrast microscopy from the globular factories formed during T3DN infection (8). From this result, we have concluded that the matrix of the viral factories is largely composed of µNS. When coexpressed with µ2, however, µNS is not present in globular inclusions but instead colocalizes with µ2 in filamentous inclusions associated with microtubules (8). The N-terminal 40 to 41 residues of µNS have been shown to be both necessary and sufficient for colocalization with µ2 (8). In more recent experiments, we have found that the RNA-binding nonstructural protein {sigma}NS, which is diffusely distributed in the cytoplasm and nucleus when expressed alone, is specifically recruited to µNS globular inclusions in a manner that requires the N-terminal 40 amino acids of µNS and is promoted by RNA (35). Other recent studies identified the S3 genome segment and its encoded protein {sigma}NS as secondary determinants of a difference in the rates of formation of viral factories in T1L- versus T3D-infected cells (33) and provided independent evidence that {sigma}NS is recruited to viral factories through its association with µNS (3, 4). Based on these observations, we have proposed a model in which the µNS matrix acts to sequester and concentrate viral proteins, viral RNAs, and possibly host factors in distinct locations within the cytoplasm, thus building the factories in which viral replication and assembly occur (8, 35).

For this report, we subjected our model to further scrutiny by using immunofluorescence (IF) microscopy to examine the subcellular locations of the three surface proteins ({lambda}1, {lambda}2, and {sigma}2) of the viral core particle in infected cells when expressed in cells in the absence of other viral proteins or when coexpressed with viral proteins µNS and/or µ2. Based on previous evidence that µNS binds to the surfaces of cores in vitro (7), we specifically hypothesized that one or more of the core surface proteins are recruited to viral factories through an association(s) with µNS. We also tested whether entering viral cores may be recruited to preformed µNS inclusions inside cells, which may have relevance for understanding how the factories initially form. These observations provide further evidence for the central but distinct roles of µNS and µ2 in recruiting viral and cellular components that are required for the assembly of cores and for replication of the genome within viral factories.


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MATERIALS AND METHODS
 
Cells and viruses. CV-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 10% fetal bovine serum (FBS) (HyClone) and 10 µg of gentamicin (Invitrogen) per ml. Reovirus strains T1L and T3D were lab stocks. The isolate of T3D in general use in our lab is designated T3DN to differentiate it from another lab's isolate (T3DC) that differs in viral factory morphology and the M1 sequence, as described previously (40). Third-passage L-cell-lysate stocks of twice plaque-purified reovirus clones were used for cell infections.

Antibodies. 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 against the µNS and µ2 proteins have been described previously (7, 8). A rabbit polyclonal antiserum against heat-inactivated whole T1L core particles (11, 26; S. Noble and M. L. Nibert, unpublished data) was used to detect {lambda}1, {lambda}2, and {sigma}2. Mouse monoclonal antibody (MAb) 7F4, which is specific for {lambda}2, was described previously (53). All antibodies were titrated to optimize signal-to-noise ratios. For some experiments, we used Alexa 488, Alexa 594, or Texas Red conjugates of µNS and µ2 antibodies that were prepared from protein A-purified IgG conjugated to fluorophores by use of kits obtained from Molecular Probes.

Mammalian expression vectors. All reovirus proteins examined in this study were expressed from genes cloned into pCI-neo (Promega). The plasmids pCI-M1(T1L) to express µ2 (40), pCI-M3(T1L) to express µNS (8), and pCI-M3(41-721) to express µNS(41-721) (8) were previously described. For expression of the {lambda}1 protein, the T1L L3 gene was excised from pFbD-L3LS2L (26) with EcoRI and was ligated to pCI-neo cut with EcoRI to generate pCI-L3(T1L). For expression of the {sigma}2 protein, the T1L S2 gene was excised from pBS-S2L (26) with EcoRV and NotI and was ligated to pCI-neo cut with NotI and XhoI after the cut XhoI site was blunt ended by treatment with the Klenow fragment of DNA polymerase I. This procedure generated pCI-S2(T1L). For expression of the {lambda}2 protein, the T1L L2 gene was excised from pPCR-Script-L2 (31) with BamHI and EcoRI and was ligated to pBluescript (Stratagene) cut with the same enzymes to generate pBS-L2(T1L). When sequenced, the subcloned T1L L2 gene was found to encode two amino acid changes relative to the published T1L L2 sequence (6), namely Phe129->Ser and Thr534->Ala. These changes were corrected by using the QuikChange site-directed mutagenesis protocol (Stratagene), and the new pBS-L2(T1L) construct was sequenced again to confirm that the encoded amino acid sequence matched that of the published T1L L2 gene. The T1L L2 gene was then removed from pBS-L2(T1L) with SpeI and EcoRI and was ligated to pCI-neo cut with NheI and EcoRI to generate pCI-L2(T1L). All enzymes were obtained from New England Biolabs unless otherwise stated.

Transfections and infections. CV-1 cells were seeded the day before infection or transfection at a density of 1.5 x 104 per cm2 in 6-well plates (9.6 cm2 per well) containing round glass coverslips (18-mm diameter). Cells were transfected with 2 µg of DNA by use of 6 µl of Lipofectamine (Invitrogen) or with 1.5 µg of DNA by use of 10 µl of Polyfect (Qiagen) according to the manufacturers' directions. Cells on coverslips were inoculated with third-passage lysates of T1L or T3D virus at 5 PFU/cell in phosphate-buffered saline (PBS) (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, pH 7.5) supplemented with 2 mM MgCl2 (PBS-MC), and the virus was allowed to adsorb to cells for 1 h at room temperature before fresh medium was added. Cells were further incubated for 18 to 24 h at 37°C before being processed for IF microscopy.

Immunoblot analysis. CV-1 cells were transfected as described above in plates without coverslips, and whole-cell lysates were collected at 24 h posttransfection (p.t.). CV-1 cells (8 x 105) were washed briefly in PBS and then were scraped into 1 ml of PBS and pelleted. The pelleted cells were resuspended in 30 µl of PBS containing protease inhibitors (Roche), lysed in sample buffer, boiled for 10 min, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Approximately 1.3 x 105 cell equivalents were analyzed per lane. Proteins were electroblotted from the gels onto nitrocellulose in 25 mM Tris-192 mM glycine, pH 8.3. The binding of antibodies was detected with alkaline phosphatase-coupled goat anti-rabbit IgG (Bio-Rad) and colorimetric reagents p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Bio-Rad).

Immunostaining and IF microscopy. Cells to be processed for IF microscopy were fixed for 10 min at room temperature in 2% paraformaldehyde or for 3 min at -20°C in 100% methanol unless otherwise stated. Cells fixed in paraformaldehyde were washed with PBS and then permeabilized and blocked in PBS containing 1% bovine serum albumin and 0.1% Triton X-100 (PBSAT). After methanol fixation, cells were incubated in PBSAT three times for 5 min at room temperature prior to incubation in primary antibody. 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 (Molecular Probes) in PBS for 5 min to counterstain cell nuclei, briefly washed in PBS, and then mounted on glass slides with Prolong (Molecular Probes). Samples were examined under a Nikon TE-300 inverted microscope equipped with phase-constrast and fluorescence optics. Images were collected as described elsewhere (40). All images were processed and prepared for presentation by using Adobe Photoshop.

Expression of µNS and infection with top-component ISVPs. CV-1 cells seeded on 18-mm-diameter coverslips as described above were transfected with 1.5 µg of pCI-M3(T1L) by use of 10 µl of Polyfect per the manufacturer's directions. After 5.5 h, the cells were washed with PBS-MC and then incubated with 100 µg of cycloheximide (Sigma) per ml in DMEM containing 10% FBS for 30 min at 37°C. Top-component infectious subvirion particles (ISVPs) were prepared by digesting purified top-component virions (1013 particles/ml) with 200 µg of {alpha}-chymotrypsin (Sigma) per ml for 10 min at 32°C, followed by quenching with 2 mM phenylmethylsulfonyl fluoride (Sigma). Each coverslip of transfected cells was incubated with 2 x 1010 top-component ISVPs and 100 µg of cycloheximide/ml in 50 µl of PBS-MC on ice for 30 min and then was placed in DMEM containing 10% FBS and 100-µg/ml cycloheximide at 37°C for 90 min before the cells were fixed and processed for IF microscopy.


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RESULTS
 
Core surface proteins localize to viral factories in reovirus-infected cells. We and others have shown that the viral core proteins {lambda}2, {lambda}3, and µ2 colocalize with µNS in viral factories in reovirus-infected cells (9, 33, 40). To examine the distributions of the remaining two core proteins, {lambda}1 and {sigma}2, we performed IF microscopy with a polyclonal antiserum raised against whole core particles (11, 26; Noble and Nibert, unpublished data). This antiserum specifically recognizes the three core surface proteins—{lambda}1, {lambda}2, and {sigma}2—by both immunoblotting (Fig. 1A) (see reference 26 for {sigma}2 data) and immunostaining of protein-expressing cells (Fig. 1C). It does not, however, recognize the two structurally minor core proteins—{lambda}3 and µ2—by either method (Fig. 1A and C; also data not shown). Viral factories were identified in this study by using antibodies derived from a µNS-specific polyclonal antiserum as previously described (8, 40). At 18 h postinfection (hpi), staining for the three core surface proteins showed that they colocalized with µNS in both the filamentous factories of reovirus T1L and the globular factories of reovirus T3DN (Fig. 1B). There was little detectable staining for these core proteins either in the nucleus or in the cytoplasm outside the factories (Fig. 1B), suggesting that all three core surface proteins were largely localized to the factories. Similarly strong colocalization of the core surface proteins and µNS was observed at 6 and 24 hpi (data not shown). Based on these and previous findings, we conclude that all three core surface proteins are concentrated in the viral factories throughout much of the infection cycle.



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  		FIG. 1. Specificity of core antiserum and distribution of reovirus core surface proteins {lambda}1, {lambda}2, and {sigma}2 and nonstructural protein µNS in infected and singly transfected cells. (A) CV-1 cells were transfected with pCI-L3(T1L) encoding {lambda}1, pCI-L2(T1L) encoding {lambda}2, pCI-L1(T3D) encoding {lambda}3, or pCI-M1(T1L) encoding µ2. At 24 h p.t., whole-cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with a core-specific rabbit polyclonal antiserum. Positions of molecular weight markers (Bio-Rad Laboratories) are indicated (in kilodaltons) to the left. Positions of the core proteins are indicated to the right; the presence of {lambda}3 and µ2 in the samples was confirmed by using other antisera in parallel (data not shown). (B) CV-1 cells were infected with T1L (top row) or T3DN (bottom row) at 5 PFU/cell, fixed at 18 hpi, and coimmunostained with the core-specific antiserum followed by goat anti-rabbit IgG conjugated to Alexa 488 (right column) or with µNS-specific rabbit IgG conjugated to Texas Red (left column). Bars, 10 µm. (C) CV-1 cells were singly transfected with pCI-L3(T1L) (top left), pCI-L2(T1L) (top right), pCI-S2(T1L) encoding {sigma}2 (bottom left), or pCI-M3(T1L) encoding µNS (bottom right) and were fixed at 18 h p.t. Samples were immunostained with the core-specific antiserum followed by goat anti-rabbit IgG conjugated to Alexa 488 (left column), {lambda}2-specific mouse MAb 7F4 followed by goat anti-mouse IgG conjugated to Alexa 488 (top right), or µNS-specific rabbit IgG conjugated to Texas Red (bottom right). Bars, 10 µm.

Core surface proteins are diffusely distributed in cells in the absence of other viral proteins. Although localized to viral factories in infected cells, the RNA-binding nonstructural protein {sigma}NS is diffusely distributed through the cytoplasm and nucleus when it is expressed in the absence of other viral proteins (4, 35). Given this observation, we investigated the distribution of core surface proteins {lambda}1, {lambda}2, and {sigma}2 when each was separately expressed. To express these proteins, we cloned their genes (L3, L2, and S2, respectively) into the mammalian expression vector pCI-neo (Promega). Each expression construct was then separately transfected into CV-1 cells, and the distribution of each protein was determined by IF microscopy. The core-specific antiserum was used to detect {lambda}1 and {sigma}2, and {lambda}2-specific MAb 7F4 (53) was used to detect {lambda}2. In this manner, we found that each of the core surface proteins was diffusely distributed through the cytoplasm and nuclei of the transfected cells (Fig. 1C). The significance of the nuclear staining is unknown and requires further study, but as noted above, significant staining for {lambda}1, {lambda}2, and {sigma}2 was not seen in the nuclei of infected cells (Fig. 1B). The diffuse distributions of {lambda}1, {lambda}2, and {sigma}2 in transfected cells (Fig. 1C) contrasted with their localization to viral factories in infected cells (Fig. 1B), suggesting that a mechanism exists in infected cells to recruit the core surface proteins to the factories.

Core surface proteins localize to globular inclusions when coexpressed with µNS. The structurally minor core protein µ2 and the RNA-binding nonstructural protein {sigma}NS localize to µNS inclusions when either of these proteins is coexpressed with µNS (8, 35). Given these observations and our findings that these proteins, as well as the core surface proteins {lambda}1, {lambda}2, and {sigma}2 (Fig. 1B), are concentrated within viral factories in infected cells, we hypothesized that µNS may also recruit and concentrate the core surface proteins within µNS inclusions in transfected cells. To address this hypothesis, we separately coexpressed each of the core surface proteins with µNS and examined the distributions of the proteins by IF microscopy using the µNS-specific antibodies and the same reagents as those used to detect each core protein as described above. The core-specific antiserum and {lambda}2-specific MAb did not cross-react with µNS inclusions in transfected cells expressing µNS in the absence of other viral proteins (data not shown). When {lambda}1, {lambda}2, and {sigma}2 were each separately coexpressed with µNS, each was concentrated in globular inclusions that colocalized with µNS (Fig. 2). The distribution of µNS was not detectably changed from that of the protein expressed in the absence of other viral proteins (8). The localization of {lambda}1, {lambda}2, and {sigma}2 to globular inclusions (Fig. 2) contrasted with their diffuse distribution in the cytoplasm and nucleus when each was expressed in the absence of µNS (Fig. 1C). We conclude from these results that µNS inclusions recruit and concentrate the core surface proteins in transfected cells.



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  		FIG. 2. Distribution of core surface proteins and µNS in doubly transfected cells. CV-1 cells were cotransfected with 1 µg of pCI-M3(T1L) encoding µNS and 1 µg of either pCI-L3(T1L) encoding {lambda}1 (top row) or pCI-L2(T1L) encoding {lambda}2 (middle row) and were fixed at 18 h p.t. Other CV-1 cells were cotransfected with 1.8 µg of pCI-M3(T1L) encoding µNS and 0.2 µg of pCI-S2(T1L) encoding {sigma}2 (bottom row) and were fixed at 18 h p.t. Cells were coimmunostained for nonstructural protein µNS (left column) and for core surface protein {lambda}1, {lambda}2, or {sigma}2 (right column) as for Fig. 1C. Bars, 10 µm.

Core surface proteins also localize to µNS(41-721) globular inclusions. A form of µNS that lacks the N-terminal 40 residues, µNS(41-721), also forms globular inclusions when expressed in the absence of other viral proteins but does not associate with either µ2 (8) (Fig. 3) or {sigma}NS upon coexpression (35). These findings may be relevant for understanding the role(s) of these proteins in infection because a smaller form of µNS, µNSC, which lacks ~5 kDa from the N terminus of µNS and is thus similar or identical to µNS(41-721), is expressed during infection (30, 55). We therefore examined the distribution of core surface proteins {lambda}1, {lambda}2, and {sigma}2 when each was separately coexpressed with µNS(41-721). We found that each of the core surface proteins colocalized in globular inclusions with µNS(41-721) (Fig. 3). The distribution of µNS(41-721) was not detectably changed from that of the protein expressed in the absence of other viral proteins (8). The findings for {lambda}1, {lambda}2, and {sigma}2 thus strongly contrasted with those for both µ2 (8) (Fig. 3) and {sigma}NS (35) and suggested that the first 40 residues of µNS are not required for association of any of the three core surface proteins with µNS inclusions.



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  		FIG. 3. Distribution of core surface proteins and µNS(41-721) in doubly transfected cells. CV-1 cells were cotransfected with 1 µg of pCI-M3(41-721) encoding µNS(41-721) and 1 µg of pCI-M1(T1L) encoding µ2 (first row), pCI-L3(T1L) encoding {lambda}1 (second row), or pCI-L2(T1L) encoding {lambda}2 (third row). Other CV-1 cells were cotransfected with 1.8 µg of pCI-M3(41-721) and 0.2 µg of pCI-S2(T1L) encoding {sigma}2 (fourth row). Cells were fixed at 18 h p.t. and coimmunostained for µNS (left column) as for Fig. 1C and for either minor core protein µ2 by using a µ2-specific rabbit IgG conjugated to Texas Red or core surface protein {lambda}1, {lambda}2, or {sigma}2 as for Fig. 1C (right column). Bars, 10 µm.

Core surface proteins localize to µ2(T1L)-µNS filamentous inclusions. The µNS protein can be redistributed from globular inclusions to microtubule-associated filamentous inclusions when coexpressed with the minor core protein µ2(T1L) (8). To determine if the {lambda}1, {lambda}2, and {sigma}2 proteins may be redistributed to filamentous inclusions along with µNS when coexpressed with µ2(T1L), we triply coexpressed these protein combinations in transfected cells and examined their distributions by IF microscopy. In all samples, µNS had a filamentous distribution (Fig. 4), similar to that observed when µNS and µ2(T1L) are coexpressed (8). Each of the core surface proteins displayed a filamentous distribution as well and colocalized with µNS (Fig. 4). We did not stain for µ2 in these experiments, but the filamentous distribution of µNS depends on its association with microtubule-associated µ2(T1L) (8), thus suggesting that µ2(T1L) colocalized with µNS and each of the core surface proteins as well. These results also provide evidence that the association of {lambda}1, {lambda}2, and {sigma}2 with µNS is specific and not due to nonspecific recruitment to globular inclusions.



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  		FIG. 4. Distribution of core surface proteins coexpressed with µ2(T1L) and µNS in triply transfected cells. CV-1 cells were cotransfected with 1 µg of pCI-M1(T1L) encoding µ2 and 1 µg of pCI-M3(T1L) encoding µNS (first row) to illustrate their colocalization. For determination of the distribution of core surface proteins, CV-1 cells were transfected with 0.67 µg of pCI-M1(T1L) and 0.67 µg of pCI-M3(T1L) along with 0.67 µg of either pCI-L3(T1L) encoding {lambda}1 (second row) or pCI-L2(T1L) encoding {lambda}2 (third row). Other CV-1 cells were cotransfected with 0.9 µg of pCI-M1(T1L), 0.9 µg of pCI-M3(T1L), and 0.2 µg of pCI-S2(T1L) encoding {sigma}2 (fourth row). Cells were fixed at 18 h p.t. and coimmunostained for major nonstructural protein µNS (left column) as for Fig. 1C and for either minor core protein µ2 or core surface protein {lambda}1, {lambda}2, or {sigma}2 (right column) as for Fig. 3. Bars, 10 µm.

Core surface proteins separately coexpressed with µ2(T1L) are diffusely distributed and exhibit only sporadic, weak association with filaments. We considered the possibility that the recruitment of core proteins to µ2(T1L)-µNS filamentous inclusions may be due to an association of the core surface proteins with µ2 rather than with µNS. To determine if {lambda}1, {lambda}2, and {sigma}2 are recruited to microtubules when coexpressed with µ2(T1L) in the absence of µNS, we separately expressed each of the core proteins along with µ2(T1L) and examined their distributions by IF microscopy using antibodies derived from a µ2-specific polyclonal antiserum, as previously described (8, 40), and either an anti-core serum or MAb 7F4. The µ2(T1L) protein showed a primarily filamentous distribution, but with some diffuse cytoplasmic staining and diffuse and punctate nuclear staining, as previously described (8, 40) (Fig. 5). In most cells, we found that {lambda}1, {lambda}2, and {sigma}2 were diffusely distributed through the cytoplasm and nuclei of the cells (Fig. 5). In occasional cells, however, each of the core surface proteins exhibited a weak filamentous distribution, which colocalized with µ2(T1L), in addition to the diffuse cytoplasmic and nuclear distribution (Fig. 5). These results suggest that µNS is required for the strong recruitment of {lambda}1, {lambda}2, and {sigma}2 to filamentous inclusions, as shown in Fig. 4, but that {lambda}1, {lambda}2, and {sigma}2 may be capable of weak associations with µ2(T1L) on filaments in the absence of µNS. We have previously reported a similarly weak association of the RNA-binding nonstructural protein {sigma}NS with the µ2(T1L) protein on filaments (35).



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  		FIG. 5. Distribution of core surface proteins coexpressed with µ2(T1L) in doubly transfected cells. CV-1 cells were cotransfected with 1 µg of pCI-M1(T1L) encoding µ2 and 1 µg of either pCI-L3(T1L) encoding {lambda}1 (first row) or pCI-L2(T1L) encoding {lambda}2 (second row). Other CV-1 cells were cotransfected with 1.8 µg of pCI-M1(T1L) and 0.2 µg of pCI-S2(T1L) encoding {sigma}2 (third row). Cells were fixed at 18 h p.t. and coimmunostained for minor core protein µ2 (left column) and for core surface protein {lambda}1, {lambda}2, or {sigma}2 (right column) as for Fig. 3. Bars, 10 µm.

Core-like particles released into the cytoplasm during entry can also be recruited to µNS globular inclusions. Recent findings indicated that viral particles similar to cores, lacking most or all copies of outer capsid proteins µ1, {sigma}1, and {sigma}3, are released into the cytoplasm during the course of cell entry by reoviruses (10). Based on previous evidence that µNS binds to the surfaces of cores in vitro (7) as well as on the new evidence in this study for associations between µNS and each of the core surface proteins, we hypothesized that core particles released into the cytoplasm during entry may be capable of binding to preformed µNS inclusions in cells. To test this hypothesis, we first expressed µNS in transfected cells at 37°C. At 5.5 h p.t., we added cycloheximide to block further protein synthesis and 30 min later exposed the cells to either ISVPs or top-component ISVPs of reovirus T1L (see Materials and Methods). After a 30-min absorption period at 4°C, the infection was allowed to proceed in the presence of cycloheximide for 90 min at 37°C, and the cells were then fixed and processed for IF microscopy. The results with ISVPs (data not shown) and top-component ISVPs (Fig. 6) were essentially identical, namely that core surface protein staining with either an anti-core serum or {lambda}2 MAb 7F4 was restricted to punctate structures in the cytoplasm. Additionally, many of these punctate structures colocalized with µNS in globular inclusions (Fig. 6). Because new protein synthesis was inhibited in this experiment, we conclude that the core surface protein staining represents intact core particles introduced into the cytoplasm (10), some of which then bound to µNS globular inclusions. The cytoplasmic core particles produced during entry by either ISVPs or top-component ISVPs of reovirus T1L showed a similar pattern of binding to µNS(41-721) globular inclusions in parallel experiments (data not shown). These findings provide strong evidence for the binding of µNS and µNS(41-721) to the surfaces of cores inside cells, consistent with previous in vitro results (7). They further suggest that µNS and perhaps µNSC as well (30, 55), which themselves are first translated from RNA transcripts produced by parental cores (primary transcriptase particles), may then bind to these same newly arrived, transcriptionally active articles, allowing them to become embedded within nascent viral factories (see Discussion for further considerations).



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  		FIG. 6. Distribution of µNS and newly arrived core particles in cells transfected with pCI-M3(T1L) and then infected with T1L top-component (TC) ISVPs. CV-1 cells were transfected with 2 µg of pCI-M3(T1L) encoding µNS, and then at 5.5 h p.t., 100 µg of cycloheximide (CHX)/ml was added to the cells. At 6 h p.t., the cells were incubated (in the presence of cycloheximide) with T1L top-component ISVPs (5,000 particles per cell) at 4°C for 30 min and then were warmed to 37°C for 90 min and fixed. (The preceding steps are summarized in the time line at the top.) Cells were immunostained either with core-specific rabbit polyclonal antiserum followed by goat anti-rabbit IgG conjugated to Alexa 488 and µNS-specific IgG conjugated to Texas Red (top row) or with {lambda}2-specific mouse MAb 7F4 followed by goat anti-mouse IgG conjugated to Alexa 488 and µNS-specific IgG conjugated to Texas Red (bottom row). Signals from individual antibodies are shown in the left and middle columns, and merged images are shown in the right column, as indicated. Bars, 10 µm. The boxed region in the merged image of the top row is magnified x4 in the inset in order to show more clearly the punctate core staining that does (yellow) or does not (green) colocalize with µNS.


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DISCUSSION
 
Several labs have reported recent progress in understanding how the cytoplasmic factories in reovirus-infected cells are formed, how different viral and cellular components are recruited to them (or vice versa), and how some differences in the factories observed between viral strains are genetically determined (3, 4, 8, 33, 35, 40). To date, including the results of this study, the network of protein-protein and protein-RNA interactions schematized in Fig. 7 has been identified. Other viral and cellular components that may be specifically recruited and play important roles in viral genome packaging, replication, and particle assembly remain to be determined. A general hypothesis that guides these studies is that the complexly structured, 130-MDa reovirus virion, including its 10 different RNA genome segments, is likely to require an organized intracellular environment in which to be assembled with high fidelity and efficiency. Another general hypothesis is that the large and complex milieu of the infected cytoplasm has compelled reoviruses to evolve signals in their various components that specify how they are recruited to and arranged within the factories. We view the viral factories as specialized organelles, and our long-range goal is to understand their genesis, structure, and functions. The factories may be related in some ways to cellular aggresomes (24, 28, 40), and thus our studies of the factories may reveal some new aspects of cellular functions as well.



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  		FIG. 7. Summary model of µNS associations with other components in viral factories. (A) Bar diagram of µNS primary sequences (residues 1 to 721) indicating known features and regions shown to be required for protein associations. It remains to be demonstrated that µNS(41-721) and µNSC are equivalent. (B) Cartoon depicting the factory of a reovirus strain, such as T1L, whose µ2 protein recruits µNS to microtubules (MT) (8, 40). Ribosomes are excluded from the factories (42, 45), so protein synthesis must occur in surrounding regions of the cytoplasm. Core surface proteins {lambda}1, {lambda}2, and {sigma}2 are recruited to the factory through association with µNS (this study). The single-stranded RNA-binding protein {sigma}NS is also recruited to the factory through association with µNS (4, 35). Core assembly, including the 10 genomic RNA segments and polymerase {lambda}3 which are not shown here, is proposed to occur within the factory but might also occur in surrounding regions of the cytoplasm as also shown in the cartoon. Cores assembled in the cytoplasm may then be recruited to the factory through association with µNS (this study). New plus-strand RNA transcripts produced by µNS-associated cores (36, 37) within the factory may be largely retained there, possibly by binding to {sigma}NS (a), which may promote their assembly into progeny particles (sponge model). Some newly produced viral transcripts, however, must be released into the surrounding cytoplasm (b) to promote ongoing viral protein synthesis. The precise role of µNSC remains unclear and is therefore not shown. The mechanism of outer capsid assembly is also unclear and therefore not shown. Although the various protein associations are shown as direct interactions, there may be unidentified intermediaries or promoting agents in one or more cases.

Reovirus core surface proteins are separately recruited to µNS factory-like inclusions: evidence for core assembly within the factories? Previously, the reovirus nonstructural protein µNS has been shown to form inclusions that are similar in morphology to viral factories (8) and to recruit the viral proteins µ2 (8) and {sigma}NS (4, 35) to those inclusions. In addition, µNS has been found attached to progeny cores in transcriptionally active particle assembly intermediates isolated from infected cells (36, 37), and these are thought to represent the secondary transcriptase particles that produce 80 to 95% of the viral transcripts during an infection (reviewed in reference 58). Lastly, recombinant µNS has been shown to bind to the surfaces of viral core particles in vitro (7). These data identify µNS as a strong candidate for recruiting additional viral components, especially the viral core surface proteins, to factories. Thus, the starting hypothesis of this study was that one or more of these proteins may be separately recruited to viral factories through a specific association with µNS. Our new results indicate that in fact each of the three core surface proteins can be independently recruited to µNS inclusions (Fig. 2).

We were unable to distinguish between assembled cores and unassembled proteins in infected cells since the available antibodies recognize core surface proteins that are both free and bound in core particles. In transfected cells, however, the individually expressed core surface proteins are not assembled into particles because the necessary companion proteins are not coexpressed (26, 56). Under these conditions, each of the core surface proteins was separately recruited to µNS inclusions. These results suggest that {lambda}1, {lambda}2, and {sigma}2 may each be recruited to viral factories in infected cells prior to assembly into cores, which is consistent with the hypothesis that core assembly occurs within the factories. However, since the expression of core proteins from either vaccinia or baculovirus vectors allows the assembly of core-like particles in the absence of µNS coexpression (26, 56), it appears likely that particles can assemble in the cytoplasm of infected cells outside the factories as well. Since the vaccinia- or baculovirus-generated core-like particles lack genomic RNA (26, 56), an interesting hypothesis is that core assembly in association with µNS-containing factories may be necessary to promote packaging of the viral genome (see below).

Reovirus nonstructural protein {sigma}NS is a single-stranded RNA-binding protein (17, 19, 21) that localizes to µNS inclusions (4, 35) through RNA-enhanced associations requiring the N termini of both µNS and {sigma}NS (35). We have proposed that {sigma}NS may recruit or retain viral mRNAs within the factories (35). Consistent with this hypothesis, we have recently obtained evidence that large amounts of newly transcribed viral RNAs localize to factories in infected cells (C. L. Miller and M. L. Nibert, unpublished data). A high concentration of viral RNAs within factories may promote their assortment and packaging into newly assembling core particles. We propose that top-component virions (50), which largely lack the viral genome (15, 16, 50), might be derived from cores that assemble in the cytoplasm and therefore fail to package the viral RNAs, whereas genome-containing virions might be derived from cores that assemble in the factories, where both the viral RNAs and core proteins are concentrated. In any case, based on evidence in this and previous studies (Fig. 1B and 6) (7), cores with or without a genome that may assemble in the cytoplasm are likely recruited to µNS-containing factories through associations of the core surface proteins with µNS.

µNS recruits different components to factories through different regions of its primary sequence: unique roles for µNSC in the viral life cycle? Previous studies have shown that the N-terminal 40 residues of µNS are required for association with two different viral proteins, µ2 and {sigma}NS (8, 35). The exact residues in µNS that determine these associations remain to be identified; however, µNS residues 1 to 13 are dispensable for association with µ2 but are required for association with {sigma}NS (35) (Fig. 7A). Moreover, µNS residues 1 to 41 are sufficient for association with µ2 (8) (Fig. 7A). The region of µNS that is sufficient for association with {sigma}NS has not been reported. In this study, we determined that residues 1 to 40 are dispensable for associations between µNS and each of the core surface proteins (Fig. 7A). Further dissection of the region(s) of µNS residues 41 to 721 involved in the associations with {lambda}1, {lambda}2, and {sigma}2 awaits future studies, but the present evidence is sufficient to conclude that different regions of µNS have evolved to promote associations with different viral proteins. We hypothesize that these associations are important not only for recruiting these proteins to the viral factories, but also for arranging them properly within the factories for viral genome replication and assembly.

An N-terminally truncated form of µNS, called µNSC, is also found in infected cells (30, 55). µNSC has been proposed to be a product of alternative translation initiation from a second in-frame AUG codon 41 codons downstream of the full-length µNS start site (34, 55). The µNS(41-721) protein expressed in this and previous studies (8, 35) is therefore believed to represent this second form of µNS. The failure of µNS(41-721) to associate with either µ2 or {sigma}NS in transfected cells (8, 35) is consistent with the hypothesis that µNS and µNSC each performs some distinct function(s) in infected cells. Previous evidence that both µNS and µNS(41-721) form factory-like inclusions (8) and new evidence that both µNS and µNS(41-721) can recruit the core surface proteins to these inclusions (Fig. 2 and 3) suggest that some functions of µNS and µNSC are overlapping. The distinguishable functions could conceivably be important at several different steps in the viral replication cycle and are the subject of ongoing studies in our lab.

The data in this report do not demonstrate whether the recruitment of each core surface protein to µNS inclusions is through a direct two-component interaction, an indirect association involving one or more RNAs or a cellular protein intermediate, or some undefined mechanism whereby µNS determines that certain proteins are shuttled to the inclusions. The recruitment clearly has some specificity, however, in that core surface proteins are recruited to µNS(41-721) inclusions (Fig. 3), whereas µ2 and {sigma}NS are not (8, 35). Further evidence for specificity is that at least one of the three reovirus outer capsid proteins is not recruited to inclusions when coexpressed with µNS (4; J. S. L. Parker, K. S. Myers, and M. L. Nibert, unpublished data).

Do entering parental core particles seed viral factories? New evidence in this study demonstrates that many of the core particles released into the cytoplasm following entry and partial uncoating (10) become bound to preformed µNS factory-like inclusions within 90 min after infection of cells that have previously expressed µNS or µNS(41-721) from an expression plasmid (Fig. 6). Thus, one model for viral factory formation is that an incoming core particle transcribes and releases the viral mRNAs, which are translated by cellular machinery in adjacent regions of the cytoplasm to yield the viral proteins, including µNS, which then binds to the adjacent core and seeds a factory. Alternatively, the newly synthesized µNS could first form a small inclusion to which the nearby core is then recruited. In either case, the µNS protein is likely to recruit other viral proteins, including {lambda}1, {lambda}2, {sigma}2, {sigma}NS, and µ2, to enlarge the nascent factory (4, 8, 35). We and others have previously noted similarities between viral factories and cellular aggresomes (8, 20, 40), structures that develop in the cytoplasm of cells overexpressing misfolded proteins (24). The two preceding hypotheses for reovirus factory formation are both similar to a seeding hypothesis proposed for the development of aggresomes (28).

If cores are embedded within a matrix of µNS that also recruits the viral RNA-binding protein {sigma}NS, some of the plus-strand transcripts that are produced and released by cores might be bound up by the {sigma}NS protein recruited to the factory (Fig. 7B). In fact, the ongoing production of new viral plus-strand RNA molecules by both parental and progeny cores in situ and the ongoing addition of µNS and {sigma}NS to the developing factories could create a sponge-like environment in which the viral plus-strand RNAs remain largely trapped within the factory. Excess transcripts could be released into the surrounding cytoplasm for translation (the factories lack ribosomes [42, 45]) when the "sponge" is saturated. In this scenario, no specificity for the viral RNAs would be required for the enrichment of viral over cellular transcripts within the factories, consistent with evidence that {sigma}NS has a nonspecific RNA-binding activity (18, 21). As a result, the viral plus-strand RNAs would be concentrated within the factories for assortment and packaging into progeny cores. In addition, because of their larger numbers of {sigma}NS-binding sites per molecule (18) and lower rates of diffusion, larger RNAs may be retained in the factories at higher frequencies than smaller ones. Since the viral plus-strand RNAs are transcribed by cores in nonequimolar amounts that are proportional to size (small > medium > large RNAs) (60), a sponge-like environment that favors the retention of larger over smaller RNAs in the factories would tend to equalize the concentrations of the 10 different transcripts. This could, in turn, promote equimolar assortment and packaging of the 10 RNAs into newly assembling cores and virions, as is thought to occur in the factories.

There are, of course, many other possible scenarios for the complex events in the reovirus life cycle that encompass transcription, translation, replication, packaging, and assembly. For example, instead of only transcripts being released from the factories as in the preceding model (Fig. 7B), some whole cores could be released to produce the transcripts for translation, whereas other cores could be retained in the factories to produce the transcripts for replication and packaging. Only a small number of released cores might be needed to generate sufficient mRNAs and proteins to fuel the viral factories, consistent with our IF microscopy evidence that the core proteins are in fact highly concentrated within the factories in infected cells (Fig. 1B). It is also important to note that the preceding model does not yet incorporate the events in outer capsid assembly that are required to complete the production of infectious virions. Further studies of the localization of reovirus RNA and protein molecules within cells are clearly needed to test these and other hypotheses relating to RNA assortment and packaging and particle assembly within reovirus-infected cells.


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ACKNOWLEDGMENTS
 
We express our sincere thanks to Elaine Freimont for lab maintenance and technical assistance and to other members of our lab for helpful discussions. We also thank Cindy Luongo for providing pPCR-Script-L2.

This work was supported by NIH grants R01 AI47904 (to M.L.N.) and K08 AI52209 (to J.S.L.P.) and by a junior faculty research grant from the Giovanni Armenise-Harvard Foundation (to M.L.N.). C.L.M. and T.J.B. received additional support, respectively, from NIH grants T32 AI07061 to the Combined Infectious Diseases Training Program at Harvard Medical School and T32 AI07245 to the Viral Infectivity Training Program at Harvard Medical School. C.D.S.P. received additional support from the Harvard College Research Program.


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FOOTNOTES
 
* Corresponding author. Mailing address for Max L. Nibert: 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: max_nibert{at}hms.harvard.edu. Present address for John S. L. Parker: James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Rd., Ithaca, NY 14853. Phone: (607) 256-5626. Fax: (607) 256-5608. E-mail: jsp7{at}cornell.edu. Back


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Journal of Virology, February 2004, p. 1882-1892, Vol. 78, No. 4
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.4.1882-1892.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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