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Nuclear Transport of Baculovirus: Revealing the Nuclear Pore Complex Passage ⇑ Shelly Au, Nelly Panté

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Nuclear Transport of Baculovirus: Revealing the Nuclear Pore Complex Passage ⇑ Shelly Au, Nelly Panté Journal of Structural Biology xxx (2011) xxx–xxx Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi Nuclear transport of baculovirus: Revealing the nuclear pore complex passage ⇑ Shelly Au, Nelly Panté Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4 article info abstract Article history: Baculoviruses are one of the largest viruses that replicate in the nucleus of their host cells. During an Available online xxxx infection the capsid, containing the DNA viral genome, is released into the cytoplasm and delivers the genome into the nucleus by a mechanism that is largely unknown. Here, we used capsids of the baculo- Keywords: virus Autographa californica multiple nucleopolyhedrovirus in combination with electron microscopy and Electron microscopy discovered this capsid crosses the NPC and enters into the nucleus intact, where it releases its genome. To Electron tomography better illustrate the existence of this capsid through the NPC in its native conformation, we reconstructed Nuclear import the nuclear import event using electron tomography. In addition, using different experimental condi- Nuclear pore complex tions, we were able to visualize the intact capsid interacting with NPC cytoplasmic filaments, as an initial Baculovirus capsid docking site, and midway through the NPC. Our data suggests the NPC central channel undergoes large- scale rearrangements to allow translocation of the intact 250-nm long baculovirus capsid. We discuss our results in the light of the hypothetical models of NPC function. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction led to progressive improvements of the three-dimensional (3D) models of the NPC, the latest with a resolution of 6 nm (Beck Cellular compartmentalization is vital, in order for the cell to et al., 2007; Frenkiel-Krispin et al., 2010). According to these studies, function efficiently. The nucleus, being a membrane-enclosed orga- the NPC consists of a membrane-embedded scaffold ring and nelle, contributes significantly to the regulation of numerous cellu- peripheral components that extend into the cytoplasm (eight lar processes. For example, by controlling the access of certain cytoplasmic filaments) and nucleus (the nuclear basket). The macromolecules to the nucleus, nuclear transport can regulate tran- NE-embedded ring has a diameter of 125 nm, is 70 nm in height, scription, DNA replication, and the cell cycle. The flow of proteins, and contains a large central channel of about 50 nm in diameter RNAs, and RNA–protein complexes (such as ribosomal subunits, (Beck et al., 2004, 2007; Frenkiel-Krispin et al., 2010; Stoffler et al., messenger ribonucleoproteins (RNPs), and splicesomal RNPs) into 1999). This central channel acts as a molecular sieve, allowing pas- and out of the nucleus at a rate of up to 1500 molecules per second sive diffusion of ions and molecules smaller than 9 nm in diameter is achieved by nuclear pore complexes (NPCs) embedded within the (or proteins smaller than 40 kDa) and selective facilitated transport nuclear envelope (NE) (Kowalczyk et al., 2011; Ribbeck and Gorlich, of larger cargos up to 39 nm in diameter (Pante and Kann, 2002). The 2001; Strambio-De-Castillia et al., 2010; Wente and Rout, 2010). selectivity is dictated by a signal (stretches of amino acids called Extensive electron microscopy (EM) studies have been carried out nuclear localization signals (NLSs) or nuclear export signals) resid- to elucidate the structure of the NPC, including studies by Ueli Aebi ing on the transported molecule, which is recognized by nuclear and colleagues using several EM techniques and atomic force transport receptors (NTRs) derived from a family of proteins known microscopy (Reviewed in Elad et al., 2009; Lim et al., 2008a,b; Maco as karyopherins or importins (Chook and Suel, 2011; Strambio- et al., 2006; Pante, 2007; Rowat et al., 2008). In particular, using De-Castillia et al., 2010; Terry and Wente, 2009; Wente and Rout, state-of-the-art cryo-electron tomography Aebi and others have 2010). The main components of the NPC are proteins called nucleopo- rins (Nups). Multiple copies of about 30 different Nups make up Abbreviations: AcMNPV, Autographa californica multiple nucleopolyhedrovirus; EM, electron microscopy; FG, phenylalanine–glycine; GV, granulovirus; LSB, low- the 120-mDa metazoan NPC (Cronshaw et al., 2002). Nups have salt buffer; MBS, modified Barth’s saline; NE, nuclear envelope; NLS, nuclear been classified into three families: integral membrane proteins, localization signal; NPCs, nuclear pore complexes; NPV, nucleopolyhedrovirus; which anchor the NPC in the NE; scaffolding Nups, which form NTRs, nuclear transport receptors; Nups, nucleoporins; RNP, ribonucleoprotein; the NPC scaffold structure; and FG-Nups, which are rich in hydro- TEM, transmission electron microscope; 3D, three-dimensional; WGA, wheat germ agglutinin. phobic phenylalanine–glycine (FG) repeat motifs (Brohawn et al., ⇑ Corresponding author. Fax: +1 604 822 2416. 2009; Tetenbaum-Novatt and Rout, 2010; Walde and Kehlenbach, E-mail address: [email protected] (N. Panté). 2010). The FG-repeat regions of these Nups presumably populate 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.11.006 Please cite this article in press as: Au, S., Panté, N. Nuclear transport of baculovirus: Revealing the nuclear pore complex passage. J. Struct. Biol. (2011), doi:10.1016/j.jsb.2011.11.006 2 S. Au, N. Panté / Journal of Structural Biology xxx (2011) xxx–xxx the central channel of the NPC and form the transport barrier of the (Agri-Food Research Centre, Summerland, B.C., Canada). Baculovi- NPC. Additionally, FG-Nups have a role in the initial docking of the ruses were purified through a continuous 15–60% (w/v) sucrose cargo to the NPC, as interactions of NTRs with peripheral FG-Nups gradient centrifugation, as described by Shoji et al. (1997) and at the NPC cytoplasmic filaments dock the cargo to the NPC (Grun- Obregon-Barboza et al. (2007) with the modifications described in wald and Singer, 2010; Hamada et al., 2011; Lowe et al., 2010; Ya- Au et al. (2010). To remove the viral envelope, purified virus was seen and Blobel, 1999). treated with 1% NP40 for 1 h at 30 °C. After this incubation, the sam- Despite considerable progress in elucidating the structure and ple was washed by centrifugation to remove excess NP40, and dia- composition of the NPC, in identifying nuclear transport signals lyzed for 24 h as described in Au et al. (2010). The successful and their receptors, and in characterizing the interaction of removal of the viral envelope and the integrity of the capsid were FG-Nups and NTRs, the molecular mechanism by which molecules evaluated by examining the capsid preparation under the EM stain- translocate through the NPC central channel remains unclear. Sev- ing with uranyl acetate, as described in Au et al. (2010). eral models have attempted to explain how interaction between Stage VI oocytes were surgically removed from narcotized X. FG-Nups and NTRs allows for efficient and selective translocation laevis, as described by Pante (2006) and Au et al. (2010). Isolated of the cargo through the NPC. Of these models, the Brownian Affin- oocytes were washed with modified Barth’s saline buffer (MBS: ity Gate/Virtual Gating model (Lim et al., 2006; Rout et al., 2000, 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 2003) and the Selective Phase model (Frey and Gorlich, 2007; Frey 0.41 mM CaCl2, 10 mM HEPES, pH 7.5) and treated with collage- et al., 2006; Ribbeck and Gorlich, 2002) are the best described; they nase (5 mg/ml) in calcium-free MBS for 1 h to defolliculate the are also the most debated. A common rationale between these two oocytes. models is that FG-containing Nups form a barrier for nuclear import, and transport occurs via association and disassociation of 2.2. Oocyte microinjection NTRs with the FG repeats. Further studies in this area resulted in alternative models such as the Reduction of Dimensionality model Injection needles were made by heating and pulling micropi- (Peters, 2005) and the more recent Forest model (Yamada et al., pettes (Microcaps; Drummond) using a micropipette puller 2010). (Inject-Matic). Microinjection of Xenopus oocytes was performed While biochemical and biophysical measurements and compu- as described in Au et al. (2010) using an oocyte microinjector tational simulations have been developed to support each model, (Inject-Matic). Oocytes were injected with 100 nl of purified capsids these models remain controversial. Direct visualization of large into the cytoplasm at the white rim separating the animal and veg- cargos crossing the NPC central channel may be a good strategy etal hemispheres. As controls, oocytes were mock injected with to test these hypothetical models. In addition, such studies will fur- 100 nl of TE (10 mM Tris, 1 mM EDTA, pH 8.7). ther substantiate the ability of NPCs in setting boundaries for cargo For time-course experiments, oocytes were microinjected with selection, and will provide a clear depiction of the overall structure capsids, incubated at room temperature in MBS for 2, 4, or 8 h, and dimensions of the NPC central channel when occupied by a and prepared for EM as described below. For some experiments, in- large cargo in transit. Because viral capsids are among the largest jected oocytes were incubated at 4 °C instead of room temperature, cargos that enter the nucleus, studies on the nuclear import of viral in order to trap the capsid at intermediate stages of nuclear import. capsids may provide important information that can be used to A biochemical inhibitor of nuclear transport was also used to test the several proposed models for NPC function. At the same depict transport-arrested capsid at the NPC. Oocytes were microin- time, these studies will enhance our knowledge about the replica- jected with the lectin wheat germ agglutinin (WGA) conjugated tion cycle of viruses.
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