JCB: Report HIV-1 remodels the nuclear pore complex Anne Monette,1,2 Nelly Panté,4 and Andrew J. Mouland1,2,3 1HIV-1 RNA Trafficking Laboratory, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Québec H3T 1E2, Canada 2Department of Medicine, Division of Experimental Medicine and 3Department of Microbiology and Immunology, McGill University, Montréal, Québec H3A 1A3, Canada 4Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada uman immunodeficiency virus type 1 (HIV-1) T cells. Infection induced extensive changes in the composi- commandeers host cell proteins and machineries tion of the NE and its associated proteins, including a re- H for its replication. Our earlier work showed that markable decrease in the abundance of Nups. Immunogold HIV-1 induced the cytoplasmic retention of nucleocyto- electron microscopy revealed the translocation of Nups plasmic shuttling and ribonucleic acid (RNA)–binding into the cytoplasm. Nup62 was identified as a component proteins. This retention is dependent on nuclear export of of purified virus, and small interfering RNA depletion the viral genomic RNA and on changes in the localization studies revealed an important role for this Nup in virus and expression level of the nucleoporin (Nup) p62 gene expression and infectivity. This detailed analysis (Nup62). To further characterize the extent of perturba- highlights the profound effects on NE composition induced tion induced by HIV-1, we performed proteomics analy- by HIV-1 infection, providing further evidence of the mag- ses of nuclear envelopes (NEs) isolated from infected nitude of viral control over the cell biology of its host. Introduction Human immunodeficiency virus type 1 (HIV-1) infection in- Schmitz et al., 2010), whereas poliovirus and cardiovirus induce duces changes in the host cell transcriptome (Giri et al., 2006; their rearrangement or degradation (Gustin and Sarnow, 2001, Li et al., 2009) and proteome (Chan et al., 2007; Ringrose et al., 2002; Bardina et al., 2009; Porter and Palmenberg, 2009). 2008; Rasheed et al., 2009). Both biochemical studies and Studies using organellar proteomics can reveal proteins genome-wide short hairpin/siRNA screens have identified nucleo- that are otherwise masked during whole cell analyses and pro- porins (Nups) as HIV-1 dependency factors that assist nuclear vide information on protein localization and function, an espe- import of the preintegration complex (PIC; Ebina et al., 2004; cially useful tool for studying a virus that usurps many cellular Goff, 2008; König et al., 2008; Kok et al., 2009; Woodward machineries (Brunet et al., 2003; Gilchrist et al., 2006). This et al., 2009; Yeung et al., 2009). Nups are also required for the work extends our previous finding that HIV-1 replication im- nuclear export of viral factors during the late stages of HIV-1 poses a blockade to the nuclear import of heterogeneous nuclear THE JOURNAL OF CELL BIOLOGY replication (Zolotukhin and Felber, 1999; Hofmann et al., 2001; RNP A1 (hnRNP A1) and its transport receptor Transportin-1 Le Rouzic et al., 2002; Kiss et al., 2003; Hutten and Kehlenbach, via alterations in the localization and abundance of Nup p62 2006; Hutten et al., 2009). Approximately 30 different Nups (Nup62; Monette et al., 2009). The persistence of Nup62 at the present in multiple copies and organized in an eightfold radial NPC core, where it authenticates passing cargo, depends on the symmetry compose nuclear pore complexes (NPCs), which stud surrounding scaffolding and anchoring Nups (Wente and Rout, and span nuclear envelopes (NEs) and act as selective barriers 2010). To determine whether the block in nuclear shuttling for the nucleocytoplasmic shuttling of macromolecules (Wente imposed by HIV-1 was limited to Nup62 expression or to the and Rout, 2010). Hepatitis B virus, herpes simplex virus, malfunctioning of other Nups, we undertook a proteomic study influenza virus, and adenovirus also use Nups to access nuclei to compare the composition of purified NEs from mock- and (Trotman et al., 2001; Copeland et al., 2009; König et al., 2010; HIV-1–infected Jurkat T cells. This has enabled the identifica- tion of 413 NE-associated host proteins, with 68% showing signi­ Correspondence to Andrew J. Mouland: [email protected] ficant changes in abundance, among which many were those Abbreviations used in this paper: DIC, differential interference contrast; emPAI, exponentially modified protein abundance index; hnRNP, heterogeneous © 2011 Monette et al. This article is distributed under the terms of an Attribution– nuclear RNP; IF, immunofluorescence; KD, knockdown; LC, liquid chromatogra- Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- phy; MS/MS, tandem mass spectrometry; NE, nuclear envelope; NPC, nuclear lication date (see http://www.rupress.org/terms). After six months it is available under a pore complex; Nup, nucleoporin; OE, overexpression; PIC, preintegration com- Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, plex; vRNA, viral genomic RNA. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 193 No. 4 619–631 www.jcb.org/cgi/doi/10.1083/jcb.201008064 JCB 619 Figure 1. Procedure taken for isolating and verifying NEs from HIV-1–infected cells. (A) A diagram depicting the architecture of NEs with associated ER, nuclear lamina, and chromatin that were purified during the isolation procedure (indicated by scissors). (B) Western analysis of proteins isolated at each step of the NE purification method using mock ( )- or minimally HIV-1 (+)–infected cells to show the elimination of plasma membrane (pr55Gag and p24), 620 JCB • VOLUME 193 • NUMBER 4 • 2011 associated with NPCs. Immunogold EM revealed that at least additional control to verify peak-infected cell populations, one Nup is dislodged from NPCs and is redirected to budding coimmunostaining for Gag and cleaved Caspase-3 revealed virions. Immunofluorescence (IF) experiments suggest that that <1% (n = 5,000) of cells was apoptotic at peak infection Nup62 is essential for viral genomic RNA (vRNA) export and (Fig. 2 B). Protein gels were stained, and gel slices were excised may take leave of NPCs as part of the growing HIV-1 vRNA– for LC-MS/MS analysis (Fig. 2 C). Mascot server–generated RNP complex, where it may ensure its replicative success dur- exponentially modified protein abundance index (emPAI) values ing viral egress, gene expression, and assembly. for individual proteins were used to calculate how HIV-1 infection affects their abundance at NEs (Table S3, last column). Globally, Results and discussion HIV-1 infection dramatically impacted NE protein composition by increasing or decreasing total protein abundance by 38 and Isolation of NEs from HIV-1–infected T cells 30%, respectively (Fig. 2 D). The classification of protein hits To define changes to NPC composition in HIV-1–infected into Gene Ontology (GO)–term biological processes, and the T cells, we used a recently published method to isolate NEs and means of their cumulative emPAI values provided evidence that accompanying NPCs, associated nuclear lamina, and contigu- proteins involved in cell division, differentiation, and DNA re- ous ER from T cells for a comparative liquid chromatography pair were less prevalent in NEs from infected cells (Fig. 2 E). (LC)/tandem mass spectrometry (MS/MS) study (Fig. 1 A; Other down-regulated processes included protein secretion, Korfali et al., 2009). Because much of the starting material is posttranslational modification, translation regulation, stress re- lost from the purification procedure, we first tested the method sponse, immune response, and nucleocytoplasmic shuttling, all for its ability to enrich NEs by collecting cellular products iso- of which might otherwise elicit an antiviral state. Conversely, lated at each step of the procedure from mock- or lowly infected proteins participating in T cell activation and vesicular traf- cells (day 3 after transfection of proviral DNA). These were ficking were up-regulated, as were nucleosomal, transcription, normalized for protein content and loaded onto SDS-PAGE RNA-binding, processing, splicing, and ribosomal proteins, gels for Western analysis, which validated the purification suggesting that HIV-1 has far-reaching effects on nuclear events procedure of NEs from cells by highlighting the gradual loss that may contribute to its replicative success. In support of this, of plasma membrane–associated viral (e.g., p24 Capsid [CA] when protein hits were organized according to subcellular com- and pr55Gag), cytoplasmic (e.g., pr55Gag and glyceraldehyde 3- partments, those known to be important for HIV-1 replication, phosphate dehydrogenase), and chromatin-binding and nuclear such as chromosomal and vesicular proteins, were found to be proteins (e.g., proliferating cell nuclear antigen and Nucleolin) up-regulated (Fig. 2 F). Importantly, proteins involved in nucleo- and the accompanying enrichment of NE, NPC, and ER pro- cytoplasmic transport were most underrepresented, and this teins Lamin B1, Nup, and Calnexin, respectively (Fig. 1 B). To was reflected by a lowered abundance of nuclear and nuclear further validate the method, again, at low infection stages (day 3) membrane–associated proteins. Our observation that HIV-1 mirrored by only two cells labeled by the anti-Gag antibody infection affects the expression of nucleocytoplasmic shuttling (Fig. 1 C, top row, first panel), products from each step of the proteins is reflected by earlier studies using CD4+ and primary procedure were analyzed by IF. The staining for NE-associated cells (Chan et al., 2007, 2009). Although Chan et al. (2007, Lamin B1 and the decrease in DAPI staining demonstrate the 2009) used whole cells for their analysis, >46% of the com- enrichment in NE proteins and the removal of the majority of mon hits are modified in the same direction by HIV-1 infection chromosomal DNA contaminants, respectively (Fig. 1 C, top (Table I). In all, we find that 54, 100, 100, and 50% of our iden- row).