Nonstructural Protein NS80 Is Crucial in Recruiting Viral Components to Form Aquareoviral Factories

Background Replication and assembly of vertebrate reoviruses occur in specific intracellular compartments known as viral factories. Recently, NS88 and NS80, the nonstructural proteins from aquareoviruses, have been proposed to share common traits with µNS from orthoreoviruses, which are involved in the formation of viral factories. Methodology/Principal Findings In this study, the NS80 characteristics and its interactions with other viral components were investigated. We observed that the NS80 structure ensured its self-aggregation and selective recruitment of viral proteins to viral factories like structures (VFLS). The minimum amino acids (aa) of NS80 required for VFLS formation included 193 aa at the C-terminal. However, this truncated protein only contained one aa coil and located in the nucleus. Its N-terminal residual regions, aa 1–55 and aa 55–85, were required for recruiting viral nonstructural protein NS38 and structural protein VP3, respectively. A conserved N-terminal region of NS38, which was responsible for the interaction with NS80, was also identified. Moreover, the minimal region of C-terminal residues, aa 506–742 (Δ505), required for NS80 self-aggregation in the cytoplasm, and aa 550–742 (Δ549), which are sufficient for recruiting viral structure proteins VP1, VP2, and VP4 were also identified. Conclusions/Significance The present study shows detailed interactions between NS80 and NS38 or other viral proteins. Sequence and structure characteristics of NS80 ensures its self-aggregation to form VFLS (either in the cytoplasm or nucleus) and recruitment of viral structural or nonstructural proteins.


Introduction
Viral factories (also termed viral inclusion bodies, viroplasm, or viromatrix) are specific intracellular matrices and physical scaffolds where multiple viral components participate in viral replication and assembly. The formation of viral factories is complex and involves a series of interactions among different types of viral components, including structural and nonstructural proteins. Many recent efforts have been dedicated to identify key factors and processes in the formation of viral factories [1][2][3][4].
Members of the family Reoviridae are known to replicate and assemble within the cytoplasmic viral factories. Reoviridae contains a large and diverse group of viruses with icosahedral symmetry but may appear spherical, and non-enveloped with one-, two-, or three-layered protein capsids surrounding the linear dsRNA segments of the viral genome. Viruses in this family are divided into 15 genera [5]. Although the aquareovirus genome (reoviruses that infects aquatic species such as fish, shellfish) comprises 11 dsRNA segments and that of orthoreovirus (reoviruses that infects mammals, birds, or reptiles) comprises 10 dsRNA segments, these two distinct members are considered the most closely related genera in Reoviridae based on multiple parameters [6], [7].
Recently, NS88, a nonstructural protein of turbot Scophthalmus maximus reovirus (SMReV) was found to share notable homology with orthoreovirus mNS. Another nonstructural protein of SMReV, NS38, has been predicted to be involved in the formation of viral inclusion bodies with NS88 [9]. In addition, NS80, another nonstructural protein of aquareovirus has also been recognized as similar to SMReV NS88 and orthoreovirus mNS that play common roles in the formation of viral factories like structures (VFLS) [29]. However, a lack of detailed investigation for the expression features and protein recruitment of NS88, NS80, and their homologs hampers our understanding on the formation of aquareoviral factories and the virus replication mechanism.
In the present study, the central role of NS80 in viral factory formation was investigated. Based on the analysis of the sequence structure, expression products (full-length or truncated), and its interactions with other viral proteins, it is possible to elucidate the role of NS80. As a nonstructural protein, it showed specific subcellular localization and recruited viral proteins via certain aa sequences.

Results
C-terminal residues of NS80 affect the formation of viral factories like structures NS80 contained two coils (residues 513-550 and 615-705, Fig. 1A) according to predictions made by the coils program [30]. To identify the NS80 domains responsible for VFLS formation, the NS80 N-terminal was deleted in a stepwise manner (Fig. 1A), fused with FLAG tag, and then inserted into pcDNA3.1 vector.
The recombinant plasmids were separately transfected into the GCO cells. The expression of each construct was identified by Western blot analysis (Fig. 1B, C, D, and E). The capacities of these constructs to form globular inclusions were analyzed by immunofluorescence with anti-FLAG antibodies. The expression of NS80-FLAG resulted in the formation of globular inclusions (NS80/FLAG in Fig. 2), which were similar to those found in viral factories. Deletion of the 216 N-terminal residues had no adverse effect on the formation of inclusions. However, the inclusions formed by NS80 truncations lacking N-terminal 283 and 373 residues (NS80-D283/FLAG and NS80-D373/FLAG in Fig. 2) formed inclusions that were less spherical and smooth compared with those formed by the intact NS80.
VFLS formed in the cytoplasm during expression of the truncated mutant NS80 (418-742) (NS80-D417/FLAG in Fig. 2). Interestingly, globular inclusions formed by both the truncations NS80 (513-742) (NS80-D512/FLAG in Fig. 2), which contained the two predicted coils, and NS80 (550-742) (NS80-D549/FLAG in Fig. 2), lacking the first coil regions, were located in the nucleus rather than in the cytoplasm. In addition, globular inclusions formed by NS80 truncations lacking N-terminal 491, 499, or 504 residues were located in the entire cell, including the cytoplasm and nucleus. Expression of truncated NS80 (615-742) (NS80-D614/FLAG in Fig. 2) containing only one predicted coil showed a diffused distribution in cells. These results suggested that the 193 C-terminal residues comprising residues 550-742 compose the minimal region required for the formation of globular inclusions. However, the 512 NS80 N-terminal residues could affect the cytoplasmic location of inclusions, and the intercoil regions (aa 550-615 of NS80) play an important role in protein aggregation and nuclear localization.
In addition, the protein (NS80-H569Q) with a histidine mutation (replaced by glutamine) at position 569 could not form inclusions (NS80-H569Q/FLAG in Fig. 2). Histidine is conserved in the NS80 intercoil region and its homologs in aquareoviruses, suggesting that it may play an important role in VFLS formation.

NS80 recruits nonstructural proteins to viral factories like structures
The nonstructural protein NS38 contains 352 aa with a molecular mass of 38 kDa and is encoded by the S9 genome segment. It has a high homology with the mammalian orthoreovirus (MRV) sNS with respect to its secondary structure and hydrophobicity. Other viral proteins are also required for the formation and function of viral factories. Interactions between NS80 and NS38 were analyzed. When pcDNA3.1/NS80-FLAG and pcDNA3.1/NS38-HA were transfected in GCO cells respectively, NS80 assembled in the cytoplasm to form VFLS as indicated by green fluorescence (Fig. 2), and NS38 was diffusely distributed in the cytoplasm as indicated by red fluorescence (Fig. 3D). When these two plasmids were cotransfected, NS80 and NS38 colocalized in VFLS in the cytoplasm (presented as yellow in the merged file in Fig. 3D), indicating that NS80 assembled in the cytoplasm and recruited NS38.
Distribution of NS80 and NS38 in virus infected cells was examined by an immunofluorescence assay. NS80 and NS38 were individually expressed in Escherichia coli BL21 (DE3) and purified for antibody preparation (Fig. 3A, B, and C). In the immunofluorescence assay, NS80 was detected by anti-NS80 rabbit serum with Alex fluor 546-conjugated goat anti-rabbit secondary antibodies (red fluorescence) and NS38 was detected by anti-NS38 mouse serum with Alex fluor 488-conjugated goat antimouse secondary antibodies (green fluorescence). As shown in Fig. 3E, NS80 was aggregated and colocalized with NS38 (presented as yellow in the merged file). The nuclei of infected cells were aggregated, a typical phenomenon of aquareovirus infection.
Results of the above studies revealed that NS80 could recruit NS38. aa sequence comparisons showed that a conserved region existed in the NS38 N-terminal (residues 20-39 of NS38) and its homologs (Fig. 4A). To determine the roles of this conserved region in interactions between NS80 and NS38, the NS38 Nterminal was deleted in a stepwise manner. The expression of truncated NS38 was detected by Western blotting (Fig. 4B). When NS80-FLAG was coexpressed with NS38-D19-HA (1-19 aa deleted), they colocalized in the cytoplasm (Fig. 4C), but NS80-FLAG could not colocalize with NS38-D39-HA (1-39 aa deleted) when they were coexpressed (Fig. 4C), indicating that the conserved 20-39 NS38 residues are important for interactions between NS38 and NS80.

NS80 recruits structural proteins to viral factories like structures
Viral factories are known as the place where viruses replicate and assemble. For this reason, viral factories interact not only with nonstructural proteins but also with structural proteins. The 11 GCRV genome segments encode 12 proteins. It has been known that NS80 could interact with NS38, but whether it could interact with the other proteins remains unclear. To identify other GCRV proteins that could interact with NS80, two tags, FLAG and HA, were used for visual localization of viral proteins. Each viral protein (except NS80) was fused with the N-terminal of HA and the resulted plasmid was cotransfected with pcDNA3.1/NS80-FLAG (Fig. 5A). Results are summarized in Fig. 5A.
To determine the NS80 aa regions responsible for interactions with these four proteins (VP1, VP2, VP3, and VP4), NS80 Nterminals were gradually deleted and the C-terminals were fused with the FLAG tag. These truncated recombinant proteins were coexpressed with VP1-HA, VP2-HA, VP3-HA, and VP4-HA, respectively.
When VP3 recombinant protein was coexpressed with NS80-D55-FLAG in the cells, they colocalized in the cytoplasm (Fig. 6C), but NS80-D85-FLAG did not colocalize with VP3 when they were coexpressed (Fig. 6C), suggesting that 56-85 NS80 residues are responsible for the interactions between NS80 and VP3.

Discussion
Different from the previous report on other reoviruses, the minimal NS80 region essential for aggregation only contained one intercoil and one coil (residues 550-742 of NS80, Fig. 2 and Table 1). It has been reported that the two coil regions of mNS are necessary for MRV viral factories formation [31][32][33]. It is also indicated as the difference between aquareovirus and other reovirus. Expression of NS80(492-742), NS80(500-742), NS80(505-742), and NS80(513-742) containing two coils and the intercoil localized into the nucleus, whereas NS80(418-742) localized in the cytoplasm. Perhaps the 418-491 NS80 residue region is important for localization in the cytoplasm. Compared with NS80(550-742), NS80(615-742) was diffusely distributed in the cytoplasm and nucleus, which indicated that the intercoil region (aa 550-615) plays a vital role in NS80 aggregation and localization. Several aspects may account for this difference in localization: (i) full-length NS80 could interact directly or indirectly with cytoplasmic compartments with its N-terminal sequence, which is responsible for cytoplasmic localization.
Although the NS80 C-terminal could interact with nuclear compartments, its interaction was weaker than that of the Nterminal. It has been reported that in MRV (strain of T1L), mNS could colocalize with m2 and then interact with microtubules in cytoplasm with the help of m2 [34]. (ii) Besides the NS80 organizational role in viral factories formation, it may also have functions in the regulation of cellular response to infection (such as immunoregulation) by interacting with nuclear components. MRV (strain T1L) m2, which has nuclear distribution, inhibits interferon signaling by accumulation of interferon regulatory factor 9 in the nucleus [35]. The mechanisms determining the importance of the  By sequence comparison and intracellular localization studies, a conserved region near the NS38 N-terminal was identified, which was important for the interactions between NS38 and NS80. This region is conserved in aquareoviruses, which hints that it could have a common role in aquareovirus factories formation. Nonstructural proteins encoded by S4 and S9 genome segments are important components in aquareovirus factories formation. It is similar to the formation of viral factories in orthoreoviruses. In MRV and ARV, viral factories are formed by nonstructural proteins mNS and sNS [36][37][38][39][40].
NS80 recruited not only nonstructural proteins but also structural proteins such as VP1, VP2, VP3, and VP4. These four proteins are viral inner core proteins. They also function as viral RNA dependent RNA polymerases, helicases, and GTPases that are needed in viral replication [41]. The fact that NS80 could completely colocalize with VP1, VP2, VP3, or VP4 reveals that its interactions with the enzymatic proteins is the strongest among the examined structural proteins. It indicates that NS80 has functions as the organization center in viral factories formation. NS80 aggregates to form the framework with NS38 and then recruits other viral proteins to participate in virus replication. The viral inclusions like structures formed by MRV mNS could also recruit viral core surface proteins [42], [43]. Interestingly, NS80 truncations (NS80-D512, NS80-D549) recruited structural proteins (VP1, VP2, and VP4) to the nucleus. This phenomenon was not  . Amino acid sequence comparison of NS38 from aquareoviruses and orthoreoviruses, and immunofluorescence analysis of key sequences in interactions between NS80 and NS38. A. Amino acids alignment of N-terminal sequences of NS38 from five aquareoviruses (GCRV, CSRV, AGCRV, SMReV and CHSRV) and its homologs belonging to orthoreoviruses (sNS in MRV or ARV). The black shaded regions indicate highly conserved residues in all viruses, while the gray shaded regions are partially conserved residues with more than 80% identity.
The key region involved in protein interactions is boxed (aa 20-39 of GCRV). B. Western blot analysis of expression products of full-length and truncated NS38 in transfected cells C. Immunofluorescence analysis of NS80 coexpressed with NS38 N-terminal truncations in transfected cells. NS38-D19 (red) colocalized with NS80 (green) but NS38-D39 (red) was not colocalized with NS80 (green), which indicated that the NS38 conserved residues, 20-39, were responsible for colocalization with NS80. The nucleus was stained by Hoechst 33342 and presented as blue. Bar = 20 mm. D. Immunofluorescence analysis of NS38 coexpressed with NS80 N-terminal truncations in transfected cells. NS80-D55 (green) was not colocalized with NS38 (red). The nucleus was stained by Hoechst 33342 and presented as blue. N-terminal residues, 1-55, of NS80 were important for interactions with NS38. Bar = 20 mm.  reported in other reoviruses, which indicated the importance of NS80 N-terminal residues and the difference between aquareovirus and other reoviruses.
The NS80 residues required for interactions with other viral proteins (VP1, VP2, VP3, VP4, and NS38) were also examined (Fig. 6). Unlike MRV mNS, which recruits only one protein l3 via indicated NS80. Red fluorescence indicated the other nine viral proteins. The nucleus was stained by Hoechst 33342 and presented as blue. NS80 colocalized with VP1, VP2, VP3, and VP4, and NS80 weakly colocalized with VP6. Bar = 20 mm. doi:10.1371/journal.pone.0063737.g005 the C-terminal region [43], NS80 recruits three proteins (VP1, VP2, and VP4) via the C-terminal region. It is difficult to accurately identify the NS80 residues responsible for interactions with VP1, VP2, or VP4. Results also showed that NS80 recruited NS38 and VP3 through different N-terminal regions. Locations of these functional domains indicate the difference between aquareovirus and other reoviruses.
Aquareovirus and orthoreovirus are considered as the most closely related genera in the family Reoviridae. Most of their proteins share a similarity of more than 20% (Table 2). Similarity levels of more than 20% in aa have been considered as an indication of common ancestry within a family [5], [6]. The key proteins for viral factories formation in these two viruses are nonstructural proteins (NS80 for GCRV, mNS for MRV). However, there are some differences in protein recruitment in viral factories formation. For example, the similarity shared by GCRV VP1/MRV l2 and GCRV VP4/MRV m2 are 28% and 27%, respectively ( Table 2). The residual regions of mNS responsible for recruiting l2 and m2 are located in the N-terminal (aa 75-85 and 20-25, respectively; Fig. 7B), but the NS80 residual regions responsible for recruiting VP1 and VP4 are located at the C-terminal end (Fig. 7A). Another difference is the minimal region needed for viral factories formation. These differences indicated the divergence between aquareovirus and orthoreovirus.
The MRV mNS-based platforms have been established in animal cells and yeast for visualizing protein interactions [44], [45]. Recently, mNS of ARV has also been used for visualizing protein interactions [40], [46]. An additional nuclear localization sequence was fused to the ARV mNS so that it could work in the nucleus. In the present study, VFLS formed by the NS80 Cterminal located in the nucleus making it possible for the NS80based protein interaction platforms to work directly in the nucleus.
Interestingly, there were at least two bands detected in Western blot analysis of NS80 expression in infected cells (Fig. 3B). The NS80 polymorphism may be because of in-frame translation. More than 20 in-frame AUG codons were found in NS80, many of which were in accordance with the Kozak's initiation consensus sequence [47]. There are two forms of mNS in MRV infected cells, mNS and mNSC. Translation of mNSC starts from an in-frame AUG codon of M3 genome segment and lacks the first 40 Nterminal residues of mNS [48]. Another possible mechanism of the polymorphism is post-translational modification. Avian orthoreovirus (ARV, another orthoreovirus) generates mNS and mNSC by post-translational cleavage [49]. The exact mechanism of NS80 polymorphism needs to be further investigated. The NS80 polymorphism may be critical to the formation of viral factories. Different NS80 lengths could meet different needs in viral infection. It could increase the specificity and efficiency of recruiting different viral proteins. Understanding NS80 could improve our knowledge about the mechanisms involved in the formation of viral factories of aquareovirus and the mechanism of antiviral research in aquaculture animals [50], [51].
In conclusion, the present study shows the detailed interactions between NS80 and NS38 or other viral proteins. NS80 sequence and structure characteristics ensure its self-aggregation to form VFLS (either in the cytoplasm or nucleus) and recruitment of virus structural or nonstructural proteins (Summary in Table 1 and Fig. 7A). It would give us new insights into the mechanisms of viral factories formation and pathogenesis of aquareoviruses.

Cells and virus
Grass carp ovaries (GCO) cell lines maintained in our laboratory were used for viral infection and transfection [52]. Cells were cultured in 199 medium supplemented with 10% fetal bovine serum at 25uC and placed in CO 2 -free system. Grass carp reovirus (GCRV) used in this study was maintained in our laboratory [8].

Plasmid constructions
Primers were designed according to GCRV sequences (Table S1).
To express a C-terminal FLAG-tagged version of NS80, GCRV genomic dsRNA was used as a template. Primers 3.1-NS80-F/R was used in reverse transcription PCR (RT-PCR). In brief, genomic dsRNA was extracted from purified viral particles using the commercial TRIzol reagent (Invitrogen), according to the protocol described by the manufacturer. cDNA was reverse transcribed in a cDNA reaction containing 5 ml 56 reaction buffer, 1 ml dNTP mix (10 mM), 1 ml recombinant ribonuclease inhibitor (20 U/ml), 1 ml M-MLV RTase (Promega). The reaction was incubated in a thermal cycler at 42uC for 1 h followed by 80uC for 5 min. PCR was performed using TaKaRa Ex Taq (TaKaRa) with the same primers. The PCR product was digested with EcoRI/BamHI and then ligated to pcDNA3.1 vector (Invitrogen) with the same enzyme treatment. The resulted plasmid was named pcDNA3.1/NS80-FLAG. The aa sequence of FLAG tag in this study was DYKDDDDK.
To generate plasmids expressing NS80 truncations with a Cterminal FLAG tag, the start codons and restriction sites were introduced at different positions in the GCRV S4 segment by PCR amplification using pcDNA3.1/NS80-FLAG as the template. Each PCR product was digested with EcoRI/BamHI and then ligated to pcDNA3.1, which had been digested with the same enzymes.
To express other GCRV proteins that fused with a C-terminal HA tag, genomic dsRNA was used as the template. RT-PCR was performed with corresponding primers of each segment. The PCR product was digested and ligated to pcDNA3.1 vector with the help of different restriction enzymes. The aa sequence of HA tag in this study was YPYDVPDYA.
To generate plasmid pcDNA3.1/NS80-H569Q-FLAG, primers 3.1-NS80-H569Q-F/R that contained mutations were used in an overlap extension PCR. Primers, enzymes, and the resulted plasmids are mentioned in Table S1. All constructed plasmids were confirmed by restriction digestion and DNA sequencing. Transfection and cell staining GCO cells were seeded onto coverslips in six-well plates before transfection. Plasmids were transfected or cotransfected into GCO cells using Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. In brief, a total of 4 mg plasmid DNA was mixed with 10 ml Lipofectamine in serum free medium. After 20-min incubation, the mixture was added to cells and incubated at 25uC. The serum free medium was replaced with 199 medium supplemented with 5% fetal bovine serum after 6 h incubation. The cells were then incubated and processed for Western blotting or immunostaining.

Prokaryotic expression, protein purification and antibody preparation
For prokaryotic NS80 and NS38 expression, fragments encoding NS80 C-terminal 342 aa and whole NS38 were amplified using primers (Table S1). The PCR products were ligated into the prokaryotic vector pET-32a or pET-28a (Novagen). The resulted recombinant plasmids, named pET32a-NS80 and pET28a-NS38, were transformed into E. coli BL21 (DE3). To express the fused protein, the bacteria were induced with 1 mM IPTG for 6 h at 37uC. The fused protein was purified using HisBind purification kit (Novagen), mixed with an equal volume of Freund's adjuvant (Sigma), and thereafter used to immunize rabbit (NS80) or mice (NS38). Anti-NS80 serum was collected after immunizing the rabbit thrice, and anti-NS38 serum was collected after immunizing mice five times.
Animal experimental procedures were conducted under the institutional guidelines of the Hubei province. The protocol was approved by the Committee of Wuhan University Center for Animal Experiment (Permit Number: SCXK 2008-0004). Surgery was performed under the anesthetic sodium pentobarbital, and all efforts were made to minimize animal suffering.

Western blotting analysis
Plasmids transfected GCO cells for NS80 or NS38 expression were collected and subjected to Western blot analysis as described previously [53]. Anti-FLAG antibodies (for NS80) and anti-HA antibodies (for NS38) were used as the primary antibody at a 1:1000 dilution followed by alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibody at a 1:1000 dilution (Vector laboratories Inc) as the secondary antibody. GCO cells infected with GCRV at an m.o.i of 0.1 or mock infected were collected and analyzed by Western blotting as described above.

Immunofluorescence microscopy observation
For immunofluorescence localization, all experiments were performed according to a previous study [53]. After 24 h incubation, transfected or infected cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were permeabilized with 0.2% Triton X-100 and then blocked in 10% normal goat serum at room temperature for 1 h. The cells were incubated with primary antibodies in 1% normal goat serum for 2 h, rinsed three times for 10 min each with phosphate buffered saline (PBS) containing 1% normal goat serum, and then incubated with secondary antibodies. Anti-FLAG antibodies (mouse anti-FLAG antibody, Santa Cruz) were used for detecting NS80-FLAG recombinant protein. Anti-HA antibodies (rabbit anti-HA antibody, Santa Cruz) were used for detecting proteins that fused with HA tag. Anti-NS80 or anti-NS38 antiserum was used as the primary antibody in infected cells at a 1:100 dilution. Alexa-546conjugated goat anti-rabbit IgG or Alexa-488-conjugated goat anti-mouse IgG at a 1:500 dilution (Invitrogen) was used as the secondary antibody. Hoechst 33342 staining was used to visualize the nucleus. The samples were examined under a Leica DM IRB fluorescence microscope (Leica microsystems Ltd.). The images were processed with Adobe Photoshop (Adobe Systems, CA).

Author Contributions
Conceived and designed the experiments: QYZ. Performed the experiments: FK LBH. Analyzed the data: FK LBH QYZ. Contributed reagents/materials/analysis tools: FK LBH. Wrote the paper: FK LBH QYZ.