View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Virology 432 (2012) 495–504 Contents lists available at SciVerse ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin Lorena Va´zquez-Iglesias 1,2, Irene Lostale´-Seijo 2, Jose´ Martı´nez-Costas, Javier Benavente n Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Farmacia, y Centro Singular de Investigacio´n en Quı´mica Biolo´gica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain article info abstract Article history: A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in Received 30 May 2012 cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the Returned to author for revisions cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian 19 June 2012 cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that Accepted 5 July 2012 sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell- Available online 23 July 2012 specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II Keywords: activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to- Avian reovirus cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a hetero- SigmaA karyon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of Intracellular distribution sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors Nuclear import CRM1-dependent export contributing to limited ARV replication in mammalian cells. Nuclear matrix & 2012 Elsevier Inc. All rights reserved. Nucleocytoplasmic shuttling Introduction infected cells. The nonstructural protein p17, which is encoded by the second cistron of the ARV S1 gene, accumulates in the Avian reoviruses (ARVs) are icosahedral nonenveloped viruses nucleoplasm of both infected and transfected cells, but is that belong to the Orthoreovirus genus of the Reoviridae family excluded from the nucleolus. This protein shuttles continuously (Mertens, 2004). They are important pathogens that cause a between the nucleus and the cytoplasm and redistributes to the variety of disease conditions in birds and important losses in cytoplasm upon inhibition of RNA polymerase II activity, suggest- poultry farming (Jones, 2000; van der Heide, 2000). Avian reovir- ing that its nucleocytoplasmic distribution is transcription depen- ions contain a double protein capsid shell of 85 nm external dent (Costas et al., 2005). On the other hand, the S2-encoded core diameter, which encapsulates the viral core (Zhang et al., 2005). protein sigmaA is present the nucleolus of ARV-infected avian The outer capsid is built by proteins muB, sigmaB and sigmaC, and cells, and has been shown to enter the nucleus via a nucleoporin- the inner shell by proteins lambdaA, lambdaC and sigmaA. The dependent nondifussional mechanism that does not require viral core contains the RNA polymerase complex (proteins lamb- added cytosolic factors or energy input (Vazquez-Iglesias et al., daB and muA) and a segmented genome formed by 10 double- 2009). These data suggest that sigmaA penetrates into the nucleus stranded RNA (dsRNA) species. Four nonstructural proteins, by itself using a process that is mechanistically different from the muNS, sigmaNS, p17 and p10, are also expressed by the ARV classical nuclear localization signal (NLS)/importin pathway. genome (reviewed in Benavente and Martinez-Costas (2007)). ARV protein sigmaA plays a structural role by clamping ARV replication and morphogenesis take place exclusively together lambdaA building blocks, which is thought to stabilize within cytoplasmic phase-dense globular structures termed viral core particles (Martinez-Costas et al., 2000; Yin et al., 2000; Zhang factories, which are initially formed by the nonstructural protein et al., 2005). Cryo-electron microscopy analysis of avian reovir- muNS (reviewed in Benavente and Martinez-Costas (2006, 2007)), ions revealed that the particle contains 150 sigmaA ‘‘nodules’’ yet two ARV proteins have been detected within the nucleus of contacting mainly lambdaA, with minor contacts formed with the lambdaC turrets and other sigmaA molecules. The results of that study further revealed that sigmaA interacts with muB proteins of n Corresponding author. Fax: þ34 881815768. the outer shell (Zhang et al., 2005). E-mail address: [email protected] (J. Benavente). On the other hand, sigmaA binds dsRNA very tightly in an 1 Current address: Departamento de Bioquı´mica, Gene´tica e Inmunologı´a, Facultad de Biologı´a, Universidad de Vigo, 36310-Vigo (Pontevedra), Spain. irreversible and sequence-independent manner (Tourı´s-Otero 2 These authors contribute equally to this work. et al., 2005; Yin et al., 2000). The crystal structure of a bacterially 0042-6822/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2012.07.007 496 L. Va´zquez-Iglesias et al. / Virology 432 (2012) 495–504 expressed recombinant sigmaA revealed that this protein binds SigmaA enters the nucleus of mammalian cells by a nucleoporin- cooperatively to dsRNA and self-assembles as helical multimers dependent nondiffusional mechanism that does not require added that cover the surface of the dsRNA (Guardado-Calvo et al., 2008). cytosolic factors or energy input Finally, two reports have provided evidence that sigmaA antag- onizes the interferon-induced cellular response against ARV by The results of a recent report revealed that sigmaA itself is able preventing the activation of the dsRNA-dependent protein kinase to reach the nucleolus of avian cells, using a nucleoporin-depen- (PKR) (Gonzalez-Lopez et al., 2003; Martinez-Costas et al., 2000). dent mechanism that does not require import receptors or energy In this study we have compared the karyophilic properties of input (Vazquez-Iglesias et al., 2009). The results shown in Fig. 2A sigmaA in avian and mammalian cells. In contrast with the results revealed that sigmaA uses a similar mechanism to target the obtained in avian cells, the protein accumulates in the nucleo- nucleoplasm of mammalian cells. Thus, our findings that sigmaA plasm, but not the nucleolus, of mammalian cells. Nucleoplasm goes to the nucleus of digitonin-permeabilized Vero cells when accumulation is due to the association of sigmaA with mammalian- the import assay is performed at 30 1C (picture 1), but not when specific factors, and is prevented by blocking RNA polymerase II performed at 4 1C (picture 2), indicates that sigmaA does not activity, which induces sigmaA efflux into the cytoplasm by a Ran- enter the nucleus by passive diffusion. This is reinforced by our and CRM1-dependent mechanism. Finally, our results further findings that MBP-sigmaA (90 kDa) is still able to reach the revealed that, contrary to p17, sigmaA does not shuttle between nucleus of digitonin-permeabilized Vero cells (Fig. 2C) and that the nuclear and cytoplasmic compartments of mammalian cells. GFP-sigmaA (73 kDa) accumulates within the nucleoplasm of transfected Vero cells (not shown). On the other hand, nuclear import of sigmaA in Vero cells requires functional nucleoporins, since incubation of digitonin-permeabilized cells with wheat Results germ agglutinin (WGA), which binds to N-acetylglucosamine residues on nucleoporins (Yoneda et al., 1987), drastically reduces SigmaA exhibits different intracellular distribution in avian and sigmaA nuclear import (picture 3). Futhermore, sigmaA was still mammalian cells able to reach the nucleus of digitonin-permeabilized cells when the cytosolic extract was omitted from the transport solution To continue with the characterization of ARV core protein (picture 4), and when the cells were incubated with the inhibitors sigmaA, we compared its intracellular distribution in cells of of the classical nuclear import pathway N-ethylmaleimide (NEM) avian and mammalian origin. For this, we used primary chicken and GMP-PNP (pictures 5 and 6, respectively), suggesting that embryo fibroblasts (CEF) and the stable chicken cell line DF1 as sigmaA penetrates into the nucleus by an importin-independent avian cells, and monkey Vero and human HeLa as representative mechanism. Finally, sigmaA was still able to target the nucleus of mammalian cell lines. First of all, we analyzed the intracellular permeabilized Vero cells when the energy source was removed sigmaA distribution in ARV-infected cells. Immunofluorescence from the transport solution and when the cells were preincubated analysis of infected avian cells at 12 h post-infection (h.p.i.) with apyrase (pictures 7 and 8, respectively), suggesting that revealed that most sigmaA colocalized with muNS in cytoplasmic sigmaA is imported into the nucleus by an energy-independent viral factories, yet a minor but significant fraction colocalized mechanism. It should be mentioned here that all experiments with fibrillarin in the nucleolus (Fig. 1A, rows 1–4). Strikingly, the with digitonin-permeabilized cells carried out in this study were intracellular distribution of sigmaA in infected mammalian cells performed