Cell Entry of Avian Reovirus Modulated by Cell-Surface Annexin A2 and Adhesion G Protein-Coupled Receptor Latrophilin-2 Triggers Src and p38 MAPK Signaling Enhancing Caveolin-1- and Dynamin 2-Dependent Endocytosis.

The specifics of cell receptor-modulated avian reovirus (ARV) entry remain unknown. By using a viral overlay protein-binding assay (VOPBA) and an in-gel digestion coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we determined that cell-surface annexin A2 (AnxA2) and adhesion G protein-coupled receptor Latrophilin-2 (ADGRL2) modulate ARV entry. Direct interaction between the ARV σC protein and AnxA2 and ADGRL2 in Vero and DF-1 cells was demonstrated in situ by proximity ligation assays. By using short hairpin RNAs (shRNAs) to silence the endogenous AnxA2 and ADGRL2 genes, ARV entry could be efficiently blocked. A significant decrease in virus yields and the intracellular specific signal for σC protein was observed in Vero cells preincubated with the specific AnxA2 and ADGRL2 monoclonal antibodies, indicating that AnxA2 and ADGRL2 are involved in modulating ARV entry. Furthermore, we found that cells pretreated with the AnxA2/S100A10 heterotetramer (A2t) inhibitor A2ti-1 suppressed ARV-mediated activation of Src and p38 mitogen-activated protein kinase (MAPK), demonstrating that Src and p38 MAPK serve as downstream molecules of cell-surface AnxA2 signaling. Our results reveal that suppression of cell-surface AnxA2 with the A2ti-1 inhibitor increased Csk-Cbp interaction, suggesting that ARV entry suppresses Cbp-mediated relocation of Csk to the membrane, thereby activating Src. Furthermore, reciprocal coimmunoprecipitation assays revealed that σC can interact with signaling molecules, lipid raft, and vimentin. The current study provides novel insights into cell-surface AnxA2- and ADGRL2-modulated cell entry of ARV which triggers Src and p38 MAPK signaling to enhance caveolin-1-, dynamin 2-, and lipid raft-dependent endocytosis. IMPORTANCE By analyzing results from VOPBA and LC-MS/MS, we have determined that cell-surface AnxA2 and ADGRL2 modulate ARV entry. After ARV binding to receptors, Src and p38 MAPK signaling were triggered and, in turn, increased the phosphorylation of caveolin-1 (Tyr14) and upregulated dynamin 2 expression to facilitate caveolin-1-mediated and dynamin 2-dependent endocytosis. In this work, we demonstrated that ARV triggers Src activation by impeding Cbp-mediated relocation of Csk to the membrane in the early stages of the life cycle. This work provides better insight into cell-surface AnxA2 and ADGRL2, which upregulate Src and p38MAPK signaling pathways to enhance ARV entry and productive infection.

Production and Purification of Candidate Subunit Vaccines by IC-Tagging Protein Encapsulation.

Particulate material is more efficient in eliciting immune responses. Here we describe the production of micro- and nanospheres formed by protein muNS-Mi from avian reoviruses, loaded with foreign epitopes for their use as vaccines.

Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion.

Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the "two compartments" activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.

Hypericin-loaded graphene oxide protects ducks against a novel duck reovirus.

Novel duck reovirus (NDRV) disease is a serious infectious disease for poultry, for which no effective therapy has been established. Therefore, development of novel antivirals against NDRV is urgently needed. In present study, we developed a complex wherein hypericin (HY), which shows broad-spectrum antiviral activity, was loaded onto graphene oxide (GO), which has a high drug-loading capacity and low cytotoxicity. The antiviral activity of the complex (GO/HY) was studied in DF-1 cells and in ducklings infected with the NDRV TH11 strain. GO/HY showed a dose-dependent inhibition of NDRV replication, which may be attributed to direct virus inactivation or inhibition of virus attachment. Western blotting and indirect immunofluorescence assay (IFA) showed markedly suppressed protein expression in GO/HY-treated NDRV-infected DF-1 cells. Moreover, GO/HY prolonged the survival time of the ducklings by reducing pathological lesions caused by the infection and inhibiting viral replication in the liver and lungs. These results suggest that GO/HY has antiviral activity against NDRV both in vitro and in vivo.

Comparative proteomic analysis revealed complex responses to classical/novel duck reovirus infections in the spleen tissue of Cairna moschata.

Duck reovirus (DRV), a member of the genus Orthoreovirus in the family Reoviridae, was first isolated from Muscovy ducks. The disease associated with DRV causes great economic losses to the duck industry. However, the responses of duck (Cairna moschata) to the classical/novel DRV (C/NDRV) infections are largely unknown. To reveal the relationship of pathogenesis and immune response, the proteomes of duck spleen cells under the control and C/NDRV infections were compared. In total, 5986 proteins were identified, of which 5389 proteins were quantified. The different accumulated proteins (DAPs) under the C/NDRV infections showed displayed various biological functions and diverse subcellular localizations. The proteins related to the serine protease system were siginificantly changed, suggesting that the activated serine protease system may play an important role under the C/NDRV infections. Furthermore, the differences in the responses to the C/NRDV infections between the duck liver and spleen tissues were compared. Only a small number of common DAPs were identified in both liver and spleen tissues, suggesting diversified pattern involved in the responses to the C/NRDV infections. However, the changes in the proteins involved in the serine protease systems were similar in both liver and spleen cells. Our data may give a comprehensive resource for investigating the responses to C/NDRV infections in ducks. SIGNIFICANCE: A newly developed MS/MS-based method involving isotopomer labels and 'tandem mass' has been applied to protein accurate quantification in current years. However, no studies on the responses of duck (Cairna moschata) spleen tissue to the classical/novel DRV (C/NDRV) infections have been performed. As a continued study of our previous report on the responses of duck liver tissue to the C/NDRV infections, the current study further compared the differences in the responses to the C/NRDV infections between the duck liver and spleen tissues. Our results will provide an opportunity to reveal the relationship of pathogenesis and immune response and basic information on the pathogenicity of C/NDRV in ducks.

Stability of local secondary structure determines selectivity of viral RNA chaperones.

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

The E3 Ubiquitin Ligase Siah-1 Suppresses Avian Reovirus Infection by Targeting p10 for Degradation.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. The ARV p10 protein, a viroporin responsible for the induction of cell syncytium formation and apoptosis, is rapidly degraded in host cells. Our previous report demonstrated that cellular lysosome-associated membrane protein 1 (LAMP-1) interacted with p10 and was involved in its degradation. However, the molecular mechanism underlying LAMP-1-mediated p10 degradation remains elusive. We report here that the E3 ubiquitin ligase seven in absentia homolog 1 (Siah-1) is critical for p10 ubiquitylation. Our data show that Siah-1 ubiquitylated p10 and targeted it for proteasome degradation. Furthermore, the ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming a multicomponent complex. Thus, LAMP-1 promotes the proteasomal degradation of p10 via interacting with both p10 and the E3 ligase Siah-1. These data establish a novel host defense mechanism where LAMP-1 serves as a scaffold for both Siah-1 and p10 that allows the E3 ligase targeting p10 for ubiquitylation and degradation to suppress ARV infection.IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe. The ARV p10 protein is a virulence factor responsible for the induction of cell syncytium formation and apoptosis and is rapidly degraded in host cells. We previously found that cellular lysosome-associated membrane protein 1 (LAMP-1) interacts with p10 and is involved in its degradation. Here we report that the E3 ubiquitin ligase seven in absentia homolog 1 (Siah-1) ubiquitylated p10 and targeted it for proteasomal degradation. Furthermore, the ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming a multicomponent complex. Thus, LAMP-1 serves as an adaptor to allow Siah-1 to target p10 for degradation, thereby suppressing ARV growth in host cells.

Suppression of Vimentin Phosphorylation by the Avian Reovirus p17 through Inhibition of CDK1 and Plk1 Impacting the G2/M Phase of the Cell Cycle.

The p17 protein of avian reovirus (ARV) causes cell cycle retardation in a variety of cell lines; however, the underlying mechanism(s) by which p17 regulates the cell cycle remains largely unknown. We demonstrate for the first time that p17 interacts with CDK1 and vimentin as revealed by reciprocal co-immunoprecipitation and GST pull-down assays. Both in vitro and in vivo studies indicated that direct interaction of p17 and CDK1/vimentin was mapped within the amino terminus (aa 1-60) of p17 and central region (aa 27-118) of CDK1/vimentin. Furthermore, p17 was found to occupy the Plk1-binding site within the vimentin, thereby blocking Plk1 recruitment to CDK1-induced vimentin phosphorylation at Ser 56. Interaction of p17 to CDK1 or vimentin interferes with CDK1-catalyzed phosphorylation of vimentin at Ser 56 and subsequently vimentin phosphorylation at Ser 82 by Plk1. Furthermore, we have identified upstream signaling pathways and cellular factor(s) targeted by p17 and found that p17 regulates inhibitory phosphorylation of CDK1 and blocks vimentin phosphorylation at Ser 56 and Ser 82. The p17-mediated inactivation of CDK1 is dependent on several mechanisms, which include direct interaction with CDK1, p17-mediated suppression of Plk1 by activating the Tpr/p53 and ATM/Chk1/PP2A pathways, and p17-mediated cdc25C degradation via an ubiquitin- proteasome pathway. Additionally, depletion of p53 with a shRNA as well as inhibition of ATM and vimentin by inhibitors diminished virus yield while Tpr and CDK1 knockdown increased virus yield. Taken together, results demonstrate that p17 suppresses both CDK1 and Plk1functions, disrupts vimentin phosphorylation, causes G2/M cell cycle arrest and thus benefits virus replication.

Avian reovirus σA and σNS proteins activate the phosphatidylinositol 3-kinase-dependent Akt signalling pathway.

The present study was conducted to identify avian reovirus (ARV) proteins that can activate the phosphatidylinositol 3-kinase (PI3K)-dependent Akt pathway. Based on ARV protein amino acid sequence analysis, σA, σNS, µA, µB and µNS were identified as putative proteins capable of mediating PI3K/Akt pathway activation. The recombinant plasmids σA-pcAGEN, σNS-pcAGEN, µA-pcAGEN, µB-pcAGEN and µNS-pcAGEN were constructed and used to transfect Vero cells, and the expression levels of the corresponding genes were quantified by immunofluorescence and Western blot analysis. Phosphorylated Akt (P-Akt) levels in the transfected cells were measured by flow cytometry and Western blot analysis. The results showed that the σA, σNS, µA, µB and µNS genes were expressed in Vero cells. σA-expressing and σNS-expressing cells had higher P-Akt levels than negative control cells, pcAGEN-expressing cells and cells designed to express other proteins (i.e., µA, µB and µNS). Pre-treatment with the PI3K inhibitor LY294002 inhibited Akt phosphorylation in σA- and σNS-expressing cells. These results indicate that the σA and σNS proteins can activate the PI3K/Akt pathway.

A critical role of LAMP-1 in avian reovirus P10 degradation associated with inhibition of apoptosis and virus release.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. The ARV p10 protein, a viroporin responsible for the induction of cell syncytium formation and apoptosis, is rapidly degraded in host cells. However, the mechanism of p10 degradation and its relevance are still unclear. We report here the identification of cellular lysosome-associated membrane protein 1 (LAMP-1) as an interaction partner of p10 by yeast two-hybrid screening, immunoprecipitation and confocal microscopy assays. We found that rapid degradation of p10 was associated with ubiquitination. Importantly, ARV p10 degradation in host cells could be completely abolished by knockdown of LAMP-1 by siRNA, indicating that LAMP-1 is required for ARV p10 degradation in host cells. In contrast, overexpression of LAMP-1 facilitated p10 degradation. Furthermore, knockdown of LAMP-1 allowed p10 accumulation, enhancing p10-induced apoptosis and viral release. Thus, LAMP-1 plays a critical role in ARV p10 degradation associated with inhibition of apoptosis and viral release.

Acoustic trapping as a generic non-contact incubation site for multiplex bead-based assays.

In this study, we show a significantly reduced assay time and a greatly increased bead recovery for a commercial Luminex-based multiplex diagnostic immunoassay by performing all liquid handling steps of the assay protocol in a non-contact acoustic trapping platform. The Luminex assay is designed for detecting antibodies in poultry serum for infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus and avian reovirus. Here, we show proof-of-concept of a microfluidic system capable of being fully automated and handling samples in a parallel format with a miniature physical footprint where the affinity beads are retained in a non-contact levitated mode in a glass capillary throughout the assay protocol. The different steps are: incubation with the serum sample, secondary antibodies and fluorescent reporters and finally washing to remove any non-specifically bound species. A Luminex 200 instrument was used for the readout. The flow rates applied to the capillary during the initial trapping event and the wash steps were optimised for maximum bead recovery, resulting in a bead recovery of 75% for the complete assay. This can be compared to a bead recovery of approximately 30% when an automatic wash station was used when the assay was performed in the conventional manual format. The time for the incubation steps for a single assay was reduced by more than 50%, without affecting assay performance, since intermediate wash steps became redundant in the continuously perfused bead trapping capillary. We analyzed seven samples, in triplicates, and we can show that the readout of the assay performed in the acoustic trap compared 100% to the control ELISAs (positive or negative readout) and resulted in comparable S/P values as the conventional manual protocol. As the acoustic trapping does not require the particles to have magnetic properties, a greater degree of freedom in selecting microparticles can be provided. In extension, this can provide an opportunity to develop cheaper and more effective microparticles.

Anti-neoplastic effect of avian reovirus σ C protein on Rous sarcoma virus-induced tumors in chicken.

This study investigated the anti-neoplastic potential of avian reovirus σC (sigma C) protein on Rous sarcoma virus-induced fibrosarcoma in chicken. The recombinant vector expressing σC protein was injected intra-tumorally into specific pathogen free chicken with fibro-sarcoma at the dose 100µg per bird, while control birds were mock-treated with 100µg of empty vector per bird. Recombinant σC protein induced apoptosis in tumors of treated birds resulting in progressive tumor regression, while similar changes were absent in tumors of mock-treated controls. The σC protein-induced apoptosis in tumors was quantified by flow cytometry and the mean level of apoptosis up to 66% was observed in treated tumors, whereas any significant level of apoptosis was absent in mock-treated controls.

Rapid detection of Mycoplasma synoviae and avian reovirus in clinical samples of poultry using multiplex PCR.

Mycoplasma synoviae and avian reovirus (ARV) are associated with several disease syndromes in poultry and cause notable global economic losses in the poultry industry. Rapid and efficient diagnostics for these avian pathogens are important not only for disease control but also for prevention of clinical disease progression. However, current diagnostic methods used for surveillance of these diseases in poultry flocks are laborious and time-consuming, and they have low sensitivity. The multiplex PCR (mPCR) developed in this study has been proven to be both sensitive and specific for simultaneous M. synoviae and ARV detection and identification in clinical samples. To evaluate the mPCR assay, the diagnostic test was applied to different clinical samples from natural and experimental M. synoviae and ARV-infected poultry. Results were compared with serologic, single PCR, and immunofluorescence analyses. Tibiotarsal articulation could be the best target for simultaneous detection of M. synoviae and ARV infection. The detection limit by visualization of mPCR-amplified products was 100 pg for both pathogens. Overall, the mPCR developed and standardized in this research is a useful tool for diagnosis and screening and for surveillance and control of M. synoviae and ARV infection in poultry flocks.

The p17 nonstructural protein of avian reovirus triggers autophagy enhancing virus replication via activation of phosphatase and tensin deleted on chromosome 10 (PTEN) and AMP-activated protein kinase (AMPK), as well as dsRNA-dependent protein kinase (PKR)/eIF2α signaling pathways.

Autophagy has been shown to facilitate replication or production of avian reovirus (ARV); nevertheless, how ARV induces autophagy remains largely unknown. Here, we demonstrate that the nonstructural protein p17 of ARV functions as an activator of autophagy. ARV-infected or p17-transfected cells present a fast and strong induction of autophagy, resulting in an increased level of autophagic proteins Beclin 1 and LC3-II. Although autophagy was suppressed by 3-methyladenine or shRNAs targeting autophagic proteins (Beclin 1, ATG7, and LC3) as well as by overexpression of Bcl-2, viral transcription, σC protein synthesis, and virus yield were all significantly reduced, suggesting a key role of autophagosomes in supporting ARV replication. Furthermore, we revealed for the first time that p17 positively regulates phosphatase and tensin deleted on chromosome 10 (PTEN), AMP-activated protein kinase (AMPK), and dsRNA dependent protein kinase RNA (PKR)/eIF2α signaling pathways, accompanied by down-regulation of Akt and mammalian target of rapamycin complex 1, thereby triggering autophagy. By using p53, PTEN, PKR, AMPK, and p17 short hairpin RNA (shRNA), activation of signaling pathways and LC3-II levels was significantly suppressed, suggesting that p17 triggers autophagy through activation of p53/PTEN, AMPK, and PKR signaling pathways. Furthermore, colocalization of LC3 with viral proteins (p17 and σC), p62 with LAMP2 and LC3 with Rab7 was observed under a fluorescence microscope. The expression level of p62 was increased at 18 h postinfection and then slightly decreased 24 h postinfection compared with mock infection and thapsigargin treatment. Furthermore, disruption of autophagosome-lysosome fusion by shRNAs targeting LAMP2 or Rab7a resulted in inhibition of viral protein synthesis and virus yield, suggesting that formation of autolysosome benefits virus replication. Taken together, our results suggest that ARV induces formation of autolysosome but does not induce complete autophagic flux.

Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin.

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

[Expression and identification of sigmaC gene of avian reovirus by baculovirus expression system].

Recombinant baculovirus containing sigmaC gene of Avian reovirus was constructed using Bac-To-Bac Baculovirus expression system, and recombinant sigmaC protein was expressed by infecting the sf9 cell with recombinant baculovirus. Firstly, sigmaC gene of Avian reovirus was cloned and inserted into donor plasmid pFastBacHTA to obtain recombinant donor plasmid pFsigmaC. Plasmid pFsigmaC was transformed into E. coli DH10Bac for integration into bacmid vector and the recombinant bacmid plasmid BacmidsigmaC was obtained. Recombinant baculovirus rBacsigmaC was obtained by transfection of the sf9 cells with BacmidsigmaC. Western blot and indirect immunofluorescence assay (IFA) were carried and the results showed that the recombinant sigmaC protein with 37 kDa molecular weight was expressed successfully.

Cell entry of avian reovirus follows a caveolin-1-mediated and dynamin-2-dependent endocytic pathway that requires activation of p38 mitogen-activated protein kinase (MAPK) and Src signaling pathways as well as microtubules and small GTPase Rab5 protein.

Very little is known about the mechanism of cell entry of avian reovirus (ARV). The aim of this study was to explore the mechanism of ARV entry and subsequent infection. Cholesterol mainly affected the early steps of the ARV life cycle, because the presence of cholesterol before and during viral adsorption greatly blocked ARV infectivity. Although we have demonstrated that ARV facilitating p38 MAPK is beneficial for virus replication, its mechanism remains unknown. Here, we show that ARV-induced phosphorylation of caveolin-1 (Tyr(14)), dynamin-2 expression, and Rac1 activation through activation of p38 MAPK and Src in the early stage of the virus life cycle is beneficial for virus entry and productive infection. The strong inhibition by dynasore, a specific inhibitor of dynamin-2, and depletion of endogenous caveolin-1 or dynamin-2 by siRNAs as well as the caveolin-1 colocalization study implicate caveolin-1-mediated and dynamin-2-dependent endocytosis as a significant avenue of ARV entry. By means of pharmacological inhibitors, dominant negative mutants, and siRNA of various cellular proteins and signaling molecules, phosphorylation of caveolin-1, dynamin-2 expression, and Rac1 activation were suppressed, suggesting that by orchestrating p38 MAPK, Src, and Rac1 signaling cascade in the target cells, ARV creates an appropriate intracellular environment facilitating virus entry and productive infection. Furthermore, disruption of microtubules, Rab5, or endosome acidification all inhibited ARV infection, suggesting that microtubules and small GTPase Rab5, which regulate transport to early endosome, are crucial for survival of ARV and that exposure of the virus to acidic pH is required for productive infection.

A versatile molecular tagging method for targeting proteins to avian reovirus muNS inclusions. Use in protein immobilization and purification.

BACKGROUND: Avian reoviruses replicate in viral factories, which are dense cytoplasmic compartments established by protein-protein interactions. The non-structural protein muNS forms the factory scaffold that attracts other viral components in a controlled fashion. To create such a three-dimensional network, muNS uses several different self-interacting domains. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have devised a strategy to identify muNS regions containing self-interacting domains, based on the capacity of muNS-derived inclusions to recruit muNS fragments. The results revealed that the muNS region consisting of residues 477-542 was recruited with the best efficiency, and this raised the idea of using this fragment as a molecular tag for delivering foreign proteins to muNS inclusions. By combining such tagging system with our previously established method for purifying muNS inclusions from baculovirus-infected insect cells, we have developed a novel protein purification protocol. CONCLUSIONS/SIGNIFICANCE: We show that our tagging and inclusion-targeting system can be a simple, versatile and efficient method for immobilizing and purifying active proteins expressed in baculovirus-infected cells. We also demonstrate that muNS inclusions can simultaneously recruit several tagged proteins, a finding which may be used to generate protein complexes and create multiepitope particulate material for immunization purposes.

IC-tagging and protein relocation to ARV muNS inclusions: a method to study protein-protein interactions in the cytoplasm or nucleus of living cells.

BACKGROUND: Characterization of protein-protein interactions is essential for understanding cellular functions. Although there are many published methods to analyze protein-protein interactions, most of them present serious limitations. In a different study we have characterized a novel avian reovirus muNS-based protein tagging and inclusion targeting method, and demonstrated its validity to purify free an immobilized protein. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a method to identify protein-protein interactions inside living eukaryotic cells (tested in primate and avian cells). When p53 was tagged with Intercoil (IC; muNS residues 477-542), it not only got integrated into muNS cytoplasmic inclusions, but also attracted its known ligand SV40 large T antigen (TAg) to these structures. We have also adapted this system to work within the cell nucleus, by creating muNS-related protein chimeras that form nuclear inclusions. We show that nuclear muNS-derived inclusions are as efficient as cytoplasmic ones in capturing IC-tagged proteins, and that the proteins targeted to nuclear inclusions are able to interact with their known ligands. CONCLUSIONS/SIGNIFICANCE: Our protein redistribution method does not present the architectural requirement of re-constructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only cell transfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus of living cells to test protein-protein interactions or to perform functional studies by protein ligand sequestration.

Avian reovirus sigmaA localizes to the nucleolus and enters the nucleus by a nonclassical energy- and carrier-independent pathway.

Avian reovirus sigmaA is a double-stranded RNA (dsRNA)-binding protein that has been shown to stabilize viral core particles and to protect the virus against the antiviral action of interferon. To continue with the characterization of this viral protein, we have investigated its intracellular distribution in avian cells. Most sigmaA accumulates into cytoplasmic viral factories of infected cells, and yet a significant fraction was detected in the nucleolus. The protein also localizes in the nucleolus of transfected cells, suggesting that nucleolar targeting is not facilitated by the viral infection or by viral factors. Assays performed in both intact cells and digitonin-permeabilized cells demonstrate that sigmaA is able to enter the nucleus via a nucleoporin-dependent nondiffusional mechanism that does not require added cytosolic factors or energy input. These results indicate that sigmaA by itself is able to penetrate into the nucleus using a process that is mechanistically different from the classical nuclear localization signal/importin pathway. On the other hand, two sigmaA arginines that are necessary for dsRNA binding are also required for nucleolar localization, suggesting that dsRNA-binding and nucleolar targeting are intimately linked properties of the viral protein.