Global Interactomics Uncovers Extensive Organellar Targeting by Zika Virus
2018; Elsevier BV; Volume: 17; Issue: 11 Linguagem: Inglês
10.1074/mcp.tir118.000800
ISSN1535-9484
AutoresÉtienne Coyaud, Charlene Ranadheera, D. Cheng, João Gonçalves, Boris J.A. Dyakov, Estelle Laurent, Jonathan St‐Germain, Laurence Pelletier, Anne‐Claude Gingras, John H. Brumell, Peter K. Kim, David Safronetz, Brian Raught,
Tópico(s)Insect symbiosis and bacterial influences
ResumoZika virus (ZIKV) is a membrane enveloped Flavivirus with a positive strand RNA genome, transmitted by Aedes mosquitoes. The geographical range of ZIKV has dramatically expanded in recent decades resulting in increasing numbers of infected individuals, and the spike in ZIKV infections has been linked to significant increases in both Guillain-Barré syndrome and microcephaly. Although a large number of host proteins have been physically and/or functionally linked to other Flaviviruses, very little is known about the virus-host protein interactions established by ZIKV. Here we map host cell protein interaction profiles for each of the ten polypeptides encoded in the ZIKV genome, generating a protein topology network comprising 3033 interactions among 1224 unique human polypeptides. The interactome is enriched in proteins with roles in polypeptide processing and quality control, vesicle trafficking, RNA processing and lipid metabolism. >60% of the network components have been previously implicated in other types of viral infections; the remaining interactors comprise hundreds of new putative ZIKV functional partners. Mining this rich data set, we highlight several examples of how ZIKV may usurp or disrupt the function of host cell organelles, and uncover an important role for peroxisomes in ZIKV infection. Zika virus (ZIKV) is a membrane enveloped Flavivirus with a positive strand RNA genome, transmitted by Aedes mosquitoes. The geographical range of ZIKV has dramatically expanded in recent decades resulting in increasing numbers of infected individuals, and the spike in ZIKV infections has been linked to significant increases in both Guillain-Barré syndrome and microcephaly. Although a large number of host proteins have been physically and/or functionally linked to other Flaviviruses, very little is known about the virus-host protein interactions established by ZIKV. Here we map host cell protein interaction profiles for each of the ten polypeptides encoded in the ZIKV genome, generating a protein topology network comprising 3033 interactions among 1224 unique human polypeptides. The interactome is enriched in proteins with roles in polypeptide processing and quality control, vesicle trafficking, RNA processing and lipid metabolism. >60% of the network components have been previously implicated in other types of viral infections; the remaining interactors comprise hundreds of new putative ZIKV functional partners. Mining this rich data set, we highlight several examples of how ZIKV may usurp or disrupt the function of host cell organelles, and uncover an important role for peroxisomes in ZIKV infection. Zika virus (ZIKV) 1The abbreviations used are:ZIKVZika virusBioIDproximity-dependent biotin identificationCBCajal bodyCRISPRclustered regularly interspersed short palindromic repeatsCcapsid proteinDENVDengue virusEnvZIKV envelope proteinGOgene ontologyIFimmunofluorescenceNS1–5ZIKV nonstructural viral proteins 1–5PreMZIKV pre-membrane/membrane proteinsh/siRNAshort hairpin/small interfering RNAUPSubiquitin-proteasome system. 1The abbreviations used are:ZIKVZika virusBioIDproximity-dependent biotin identificationCBCajal bodyCRISPRclustered regularly interspersed short palindromic repeatsCcapsid proteinDENVDengue virusEnvZIKV envelope proteinGOgene ontologyIFimmunofluorescenceNS1–5ZIKV nonstructural viral proteins 1–5PreMZIKV pre-membrane/membrane proteinsh/siRNAshort hairpin/small interfering RNAUPSubiquitin-proteasome system. is a mosquito-borne (Aedes aegypti and Ae. Albopictus) single-stranded positive sense RNA arbovirus of the Flaviviridae family (1Petersen L.R. Jamieson D.J. Powers A.M. Honein M.A. Zika Virus.N. Engl. J. Med. 2016; 374: 1552-1563Crossref PubMed Scopus (879) Google Scholar, 2Chen H.L. Tang R.B. Why Zika virus infection has become a public health concern?.J. Chin. Med. Assoc. 2016; 79: 174-178Crossref PubMed Scopus (26) Google Scholar). Named after its discovery in the Zika forest of Uganda in 1947 (3Dick G.W. Kitchen S.F. Haddow A.J. Zika virus. I. Isolations and serological specificity.Trans. R. Soc. Trop. Med. Hyg. 1952; 46: 509-520Abstract Full Text PDF PubMed Scopus (1841) Google Scholar), ZIKV remained confined to Africa until recent outbreaks in Micronesia (2007) (4Duffy M.R. Chen T.H. Hancock W.T. Powers A.M. Kool J.L. Lanciotti R.S. Pretrick M. Marfel M. Holzbauer S. Dubray C. Guillaumot L. Griggs A. Bel M. Lambert A.J. Laven J. Kosoy O. Panella A. Biggerstaff B.J. Fischer M. Hayes E.B. Zika virus outbreak on Yap Island, Federated States of Micronesia.N. Engl. J. Med. 2009; 360: 2536-2543Crossref PubMed Scopus (2158) Google Scholar), French Polynesia (2013) (5Cao-Lormeau V.M. Roche C. Teissier A. Robin E. Berry A.L. Mallet H.P. Sall A.A. Musso D. Zika virus, French polynesia, South pacific, 2013.Emerg. Infect. Dis. 2014; 20: 1085-1086Crossref PubMed Scopus (55) Google Scholar), and South America (2015–2016) (6Zanluca C. Melo V.C. Mosimann A.L. Santos G.I. Santos C.N. Luz K. First report of autochthonous transmission of Zika virus in Brazil.Mem. Inst. Oswaldo Cruz. 2015; 110: 569-572Crossref PubMed Scopus (838) Google Scholar, 7Campos G.S. Bandeira A.C. Sardi S.I. Zika Virus Outbreak, Bahia, Brazil.Emerg. Infect. Dis. 2015; 21: 1885-1886Crossref PubMed Scopus (824) Google Scholar). Local transmission of ZIKV has now been reported in >50 countries (8Poland G.A. Kennedy R.B. Ovsyannikova I.G. Palacios R. Ho P.L. Kalil J. Development of vaccines against Zika virus.Lancet Infect Dis. 2018; 18: e211-e219Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Zika virus proximity-dependent biotin identification Cajal body clustered regularly interspersed short palindromic repeats capsid protein Dengue virus ZIKV envelope protein gene ontology immunofluorescence ZIKV nonstructural viral proteins 1–5 ZIKV pre-membrane/membrane protein short hairpin/small interfering RNA ubiquitin-proteasome system. Zika virus proximity-dependent biotin identification Cajal body clustered regularly interspersed short palindromic repeats capsid protein Dengue virus ZIKV envelope protein gene ontology immunofluorescence ZIKV nonstructural viral proteins 1–5 ZIKV pre-membrane/membrane protein short hairpin/small interfering RNA ubiquitin-proteasome system. Most ZIKV infections are asymptomatic, though ∼20% of infected individuals develop flu-like symptoms (rash, fever, joint pain) that resolve after a few days. Notably, however, ZIKV can infect human neural progenitors and post-mitotic neurons (9Tang H. Hammack C. Ogden S.C. Wen Z. Qian X. Li Y. Yao B. Shin J. Zhang F. Lee E.M. Christian K.M. Didier R.A. Jin P. Song H. Ming G.L. Zika virus infects human cortical neural progenitors and attenuates their growth.Cell Stem Cell. 2016; 18: 587-590Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 10Lin M.Y. Wang Y.L. Wu W.L. Wolseley V. Tsai M.T. Radic V. Thornton M.E. Grubbs B.H. Chow R.H. Huang I.C. Zika virus infects intermediate progenitor cells and post-mitotic committed neurons in human fetal brain tissues.Sci. Rep. 2017; 7: 14883Crossref PubMed Scopus (23) Google Scholar). Consistent with these observations, recent ZIKV outbreaks have been linked to significant increases in Guillain-Barré syndrome ((11Gatherer D. Kohl A. Zika virus: a previously slow pandemic spreads rapidly through the Americas.J. Gen. Virol. 2016; 97: 269-273Crossref PubMed Scopus (211) Google Scholar) an autoimmune disorder affecting neuromuscular function) and microcephaly (12Blazquez A.B. Saiz J.C. Neurological manifestations of Zika virus infection.World J. Virol. 2016; 5: 135-143Crossref PubMed Google Scholar, 13Li C. Xu D. Ye Q. Hong S. Jiang Y. Liu X. Zhang N. Shi L. Qin C.F. Xu Z. Zika virus disrupts neural progenitor development and leads to microcephaly in mice.Cell Stem Cell. 2016; 19: 120-126Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 14Devhare P. Meyer K. Steele R. Ray R.B. Ray R. Zika virus infection dysregulates human neural stem cell growth and inhibits differentiation into neuroprogenitor cells.Cell Death Dis. 2017; 8: e3106Crossref PubMed Scopus (50) Google Scholar, 15Mlakar J. Korva M. Tul N. Popovic M. Poljsak-Prijatelj M. Mraz J. Kolenc M. Resman Rus K. Vesnaver Vipotnik T. Fabjan Vodusek V. Vizjak A. Pizem J. Petrovec M. Avsic Zupanc T. Zika virus associated with microcephaly.N. Engl. J. Med. 2016; 374: 951-958Crossref PubMed Scopus (1850) Google Scholar), leading the WHO to declare a Public Health Emergency of International Concern. To date, no antiviral drugs or vaccines are available for the treatment or prevention of ZIKV infection. Phosphatidylserine receptors of the TIM (T-cell immunoglobulin and mucin domain) and TAM (TYRO, AXL, MER) families are candidate ZIKV receptors (16Merfeld E. Ben-Avi L. Kennon M. Cerveny K.L. Potential mechanisms of Zika-linked microcephaly.Wiley Interdiscip. Rev. Dev. Biol. 2017; 6: e273Crossref Scopus (25) Google Scholar, 17Perera-Lecoin M. Meertens L. Carnec X. Amara A. Flavivirus entry receptors: an update.Viruses. 2013; 6: 69-88Crossref PubMed Scopus (213) Google Scholar). Following receptor binding, virus endocytosis and vesicle acidification, the 10.7 kb RNA genome is released into the host cell cytoplasm. A single polyprotein is translated from this RNA, then proteolytically cleaved into three structural (Capsid: C; precursor membrane protein: PreM; Env: Envelope) and seven nonstructural (NS) polypeptides (NS1, 2A, 2B, 3, 4A, 4B, and 5) (18Kuno G. Chang G.J. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.Arch. Virol. 2007; 152: 687-696Crossref PubMed Scopus (378) Google Scholar, 19Shankar A. Patil A.A. Skariyachan S. Recent perspectives on genome, transmission, clinical manifestation, diagnosis, therapeutic strategies, vaccine developments, and challenges of Zika virus research.Front. Microbiol. 2017; 8: 1761Crossref PubMed Scopus (14) Google Scholar). Viral particle assembly occurs in the endoplasmic reticulum, following a dramatic reorganization of host endomembranes (reviewed in (20Wang A. Thurmond S. Islas L. Hui K. Hai R. Zika virus genome biology and molecular pathogenesis.Emerg. Microbes Infect. 2017; 6: e13Crossref PubMed Scopus (77) Google Scholar)). sh/siRNA and CRISPR-Cas9-based screens have identified several host factors required for ZIKV infection and replication (21Savidis G. McDougall W.M. Meraner P. Perreira J.M. Portmann J.M. Trincucci G. John S.P. Aker A.M. Renzette N. Robbins D.R. Guo Z. Green S. Kowalik T.F. Brass A.L. Identification of Zika virus and Dengue virus dependency factors using functional genomics.Cell Rep. 2016; 16: 232-246Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 22Marceau C.D. Puschnik A.S. Majzoub K. Ooi Y.S. Brewer S.M. Fuchs G. Swaminathan K. Mata M.A. Elias J.E. Sarnow P. Carette J.E. Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.Nature. 2016; 535: 159-163Crossref PubMed Scopus (266) Google Scholar, 23Yasunaga A. Hanna S.L. Li J. Cho H. Rose P.P. Spiridigliozzi A. Gold B. Diamond M.S. Cherry S. Genome-wide RNAi screen identifies broadly-acting host factors that inhibit arbovirus infection.PLoS Pathog. 2014; 10: e1003914Crossref PubMed Scopus (69) Google Scholar). Importantly, however, very little is known regarding individual ZIKV protein - host protein interactions. Here, we present a BioID/IP-MS ZIKV-host interactome. Overall, the interactome is significantly enriched in proteins associated with polypeptide processing and quality control, vesicle trafficking, RNA processing machinery and proteins linked to lipid metabolism. Notably, the data set also highlights extensive organellar targeting by ZIKV proteins. For example, we report that: (1) the ZIKV Capsid protein is targeted to a variety of host endomembrane locations and can "remodel" nuclear/ER membranes; (2) the NS5 helicase protein is targeted to, and partially disrupts, Cajal Bodies; (3) the NS3-NS2B protease interacts with the HOPS/CORVET complex, and modifies lysosome volume, and; (4) NS2A is specifically targeted to peroxisomal membranes. Consistent with this observation, we demonstrate that peroxisome function is linked to efficient ZIKV replication in culture. Together, the ZIKV-host interactome thus provides a valuable resource for better understanding ZIKV biology and identifies numerous virus-host protein interactions that could be targeted in antiviral therapeutic strategies. Both BioID and anti-Flag IP-MS analyses were performed on each of the ten ZIKV polypeptides, expressed individually in HEK293 T-REx cells, fused either with an N-terminal FlagBirA* or a C-terminal BirA*Flag epitope tag. MS samples prepared from two biological replicates were each analyzed twice (i.e. four MS runs) on a Thermo Q-Exactive HF quadrupole-Orbitrap mass spectrometer. For each prey protein, all four sample runs were compared against the two highest peptide counts among 16 control samples, prepared from HEK293 T-REx cells expressing the FlagBirA* tag alone (see below). Protein IDs with at least two unique peptides and an iProphet score >0.9 (corresponding to ∼1% FDR) were defined as high-confidence interactors, when their Bayesian False Discovery Rate (BFDR) assigned by SAINT Express (v3.6) was below 1% (supplemental Table S1). Additional details provided below. Individual ZIKV polypeptide sequences were generated by gene synthesis (BioBasics, Markham, ON, Canada; based on Genbank sequence ANO46305.1, and cleavage sites reported in (18Kuno G. Chang G.J. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.Arch. Virol. 2007; 152: 687-696Crossref PubMed Scopus (378) Google Scholar); full sequence information available on Prohits-web.lunenfeld.ca), and subcloned using AscI/NotI sites into pcDNA5.1 FRT/TO FlagBirA*- (N-terminal tagging), FRT/TO -BirA*Flag (C-terminal tagging), pcDNA3 GFP-, or pcDNA3 mCherry- plasmids. FAM134B was amplified with Q5 polymerase (NEB, Ipswich, MA) from HEK293 cDNA, with the following primers: KpnI-Fwd: TATAGGTACCATGGCGAGCCCGGCGCCTCCGG and AscI_Rev AAGGCGCGCCTTACTTGTCGTCATCGTCTTTGTAGTCGGCATGGCCTCCCAGCA GATTTG, and inserted into pcDNA3. Epitope-tagged ZIKV coding vectors were transfected with PolyJet (Signagen, Rockville, MD), and expressed in human Flp-In T-REx 293 cells (Invitrogen, Carlsbad, CA) or HeLa cells (as described in (24Coyaud E. Mis M. Laurent E.M. Dunham W.H. Couzens A.L. Robitaille M. Gingras A.C. Angers S. Raught B. BioID-based identification of Skp Cullin F-box (SCF)beta-TrCP1/2 E3 ligase substrates.Mol. Cell. Proteomics. 2015; 14: 1781-1795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar)). Protein expression was induced by adding 1 μg/ml tetracycline to the culture medium (DMEM, 10% fetal calf serum, Gibco, Waltham, MA) for 24 h. 293 T-REx cells were maintained at 37 °C in DMEM supplemented with 10% fetal bovine serum, 10 mm HEPES (pH 8.0) and 1% penicillin-streptomycin. HeLa cells were cultured under the same conditions. Human fibroblasts were cultured in DMEM with l-glutamine (Wisent, St-Bruno, QC, Canada) supplemented with 10% fetal bovine serum (Gibco) at 37 °C in humidified air with 5% CO2. Vero cells were cultured in MEM with l-glutamine supplemented with 5% fetal bovine serum at 37 °C and 5% CO2. ZIKV (ZIKV/Homo sapiens/PRI/PRVABC59/2015, Genbank accession no. KX087101.2) was amplified in Vero cells using MEM supplemented with l-glutamine and 1% fetal bovine serum. Plasmids were transfected using Lipofectamine-2000 (Invitrogen) following the manufacturer's instructions. Cells at 70% confluence were rinsed with PBS, and virus diluted in growth medium supplemented with l-glutamine and 1% FBS. Virus inoculum was removed after 1 h and replaced with fresh media. Cells and supernatants were harvested at the indicated times post-infection for determination of TCID50. 5 × 150 cm2 dishes of subconfluent (80%) 293 T-REx cells expressing the protein of interest were scraped into PBS, pooled, washed twice in 10 ml PBS, and collected by centrifugation at 1000 × g for 5 min at 4 °C. Cell pellets were stored at −80 °C until lysis. The cell pellet was weighed, and 1:4 pellet weight/lysis buffer (by volume) was added. Lysis buffer consisted of 50 mm HEPES-NaOH (pH 8.0), 100 mm KCl, 2 mm EDTA, 0.1% Nonidet P-40, 10% glycerol, 1 mm PMSF, 1 mm DTT, and 1:500 protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO). On resuspension, cells were incubated on ice for 10 min, subjected to one additional freeze-thaw cycle, then centrifuged at 27,000 × g for 20 min at 4 °C. The supernatant was transferred to a fresh 15 ml conical tube, and 1:1000 turbonuclease (BioVision, Milpitas, CA) plus 30 μl packed, pre-equilibrated Flag-M2 agarose beads (Sigma-Aldrich) were added. The mixture was incubated for 2 h at 4 °C with end-over-end rotation. Beads were pelleted by centrifugation at 1000 × g for 1 min and transferred with 1 ml of lysis buffer to a fresh centrifuge tube. Beads were washed once with 1 ml lysis buffer and twice with 1 ml ammonium bicarbonate (ammbic) rinsing buffer (50 mm ammbic, pH 8.0, 75 mm KCl). Elution was performed by incubating the beads with 150 μl of 125 mm ammonium hydroxide (pH ∼11). The elution step was repeated twice, and the combined eluate centrifuged at 15,000 × g for 1 min, transferred to a fresh centrifuge tube, and lyophilized. Cell pellets were resuspended in 10 ml of lysis buffer (50 mm Tris-HCl pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 0.1% SDS, 1:500 protease inhibitor mixture (Sigma-Aldrich), 1:1000 turbonuclease), incubated on an end-over-end rotator at 4 °C for 1 h, briefly sonicated to disrupt any visible aggregates, then centrifuged at 16,000 × g for 30 min at 4 °C. The supernatant was transferred to a fresh 15 ml conical tube, 30 μl of packed, pre-equilibrated streptavidin-Sepharose beads (GE, Boston, MA) were added, and the mixture incubated for 3 h at 4 °C with end-over-end rotation. Beads were pelleted by centrifugation at 2000 rpm for 2 min and transferred with 1 ml of lysis buffer to a fresh Eppendorf tube. Beads were washed once with 1 ml lysis buffer and twice with 1 ml of 50 mm ammbic (pH 8.3). Beads were transferred in ammbic to a fresh centrifuge tube and washed two more times with 1 ml ammbic buffer. Tryptic digestion was performed by incubating the beads with 1 μg MS grade TPCK trypsin (Promega, Madison, WI) dissolved in 200 μl of 50 mm ammbic (pH 8.3) overnight at 37 °C. The following morning, an additional 0.5 μg trypsin was added, and the beads incubated for 2 h at 37 °C. Beads were pelleted by centrifugation at 2000 × g for 2 min, and the supernatant was transferred to a fresh Eppendorf tube. Beads were washed twice with 150 μl of 50 mm ammonium bicarbonate, and washes pooled with the eluate. The sample was lyophilized and resuspended in buffer A (0.1% formic acid). One-fifth of the sample was analyzed per MS run. High performance liquid chromatography was conducted using a 2 cm pre-column (Acclaim PepMap 50 mm × 100 μm inner diameter (ID)) and 50 cm analytical column (Acclaim PepMap, 500 mm × 75 μm ID; C18; 2 um; 100 Å, Thermo Fisher Scientific, Waltham, MA), running a 120 min reversed-phase buffer gradient at 225 nl/min on a Proxeon EASY-nLC 1000 pump in-line with a Thermo Q-Exactive HF quadrupole-Orbitrap mass spectrometer. A parent ion scan was performed using a resolving power of 60,000, then up to the twenty most intense peaks were selected for MS/MS (minimum ion count of 1000 for activation) using higher energy collision induced dissociation (HCD) fragmentation. Dynamic exclusion was activated such that MS/MS of the same m/z (within a range of 10 ppm; exclusion list size = 500) detected twice within 5 s were excluded from analysis for 15 s. For protein identification, Thermo .RAW files were converted to the .mzXML format using ProteoWizard (v3.0.10800; 4/27/2017) (25Kessner D. Chambers M. Burke R. Agus D. Mallick P. ProteoWizard: open source software for rapid proteomics tools development.Bioinformatics. 2008; 24: 2534-2536Crossref PubMed Scopus (1218) Google Scholar). Using the Trans-Proteomic Pipeline (TPP v4.7 Polar Vortex rev 1, linux build 201705011551) converted files were searched using X!Tandem (Jackhammer TPP 2013.06.15.1) (26Craig R. Beavis R.C. TANDEM: matching proteins with tandem mass spectra.Bioinformatics. 2004; 20: 1466-1467Crossref PubMed Scopus (1987) Google Scholar) and Comet (2014.02 rev. 2) (27Eng J.K. Jahan T.A. Hoopmann M.R. Comet: an open-source MS/MS sequence database search tool.Proteomics. 2013; 13: 22-24Crossref PubMed Scopus (781) Google Scholar) against the Human RefSeq Version 45 database (containing 36113 entries). Search parameters specified a parent ion mass tolerance of 10 ppm, and an MS/MS fragment ion tolerance of 0.4 Da, with up to 2 missed cleavages allowed for trypsin. No fixed modifications were used. Variable modifications of [email protected] and W, [email protected] and W, [email protected] terminus, and [email protected] and Q were allowed. Proteins identified with an iProphet cut-off of 0.9 (corresponding to ≤1% FDR) and at least two unique peptides were analyzed with SAINT Express v.3.6. Control runs (16 runs for BioID; 16 runs for IP-MS; all from cells expressing the FlagBirA* epitope tag only) were collapsed to the four highest spectral counts for each prey, and high confidence interactors were defined as those with BFDR≤0.01. Each bait protein was analyzed with both an N- and C-terminal BirA* tag. Two biological replicates (i.e. separate transfections) were each subjected to two MS runs (two technical replicates). Each of the ten ZIKV bait proteins was analyzed using both BioID and Flag IP, for a total of 160 MS runs. Average R2 between technical replicates: 0.976. Average R2 between biological replicates: 0.888 (supplemental Table S1). SAINT data were imported into Cytoscape 3.4 (http://www.cytoscape.org). All network files and parameters are available at http://prohits-web.lunenfeld.ca/. Transiently transfected HeLa cells expressing GFP- or FlagBirA*-tagged proteins were grown on coverslips, fixed with 4% paraformaldehyde for 15 min, and washed in PBS with 0.1% Triton X-100. Cells were blocked in 5% bovine serum albumin (BSA) in PBS for 30 min before incubating in the indicated primary antibodies for 1 h at RT. Primary antibodies were used at the following concentrations: anti-FLAG M2 (1:500; Sigma-Aldrich), anti-CANX C5C9 (1:50; Cell Signaling Technologies, Danvers, MA), anti-Lamin B1 (1:1000; Abcam 16048), anti-COIL (1:1000; Abcam 11822), anti-P4HB (1:1000; Stressgen, Victoria, BC, Canada, SPA-891), anti-Fibrillarin (1:100; Abcam 5821). After removing the antibody solution, cells were washed once and incubated for 1 h with anti-mouse or anti-rabbit Alexa (488, 594, or 647)-conjugated secondary antibodies. In some cases, Streptavidin-Alexa594 (1:5000, Invitrogen) or BODIPY 493/503 (1:100, Invitrogen) were added with secondary antibodies. Cells were washed twice with PBS, then with 1 μg/ml of 4′,6-diamidino-2- phenylindole (DAPI) in PBS for 5 min. After washing with PBS three more times for 5 min each, coverslips were mounted with ProLong Gold Antifade (Thermo Fischer Scientific). Cells were imaged using PlanApo 60X oil lens, NA 1.40 on an Olympus FV1000 confocal microscope (zoom factor between 3–5; Olympus America, Melville, NY). Images were processed using the Volocity Viewer v.6 and assembled using Adobe Illustrator CS6 (Adobe Systems Inc.). HeLa cells were transfected with the FlagBirA*-NS5 vector, incubated 24 h, then incubated an additional 24 h on coverslips with biotin (50 μm). Cells were fixed and permeabilized as above, then stained with anti-COIL and Streptavidin AF594 (as a reporter of positive FlagBirA*-NS5 cells). Cajal body (CB) volumes (red objects gated between 0.1–5 μm3) were quantified using the Volocity 6.0 measurement tool. In each experiment, CB volumes (COIL-positive nuclear bodies) were quantified in DAPI positive objects (gated between 100–500 μm3) of the NS5-positive gated population versus the NS5 low/negative gated population. Data (raw data supplemental Table S4) of 25 stitched fields for each of the two independent experiments were merged and plotted using Excel. For calculation of the number of CB/cell, three nonoverlapping fields per experiment were used, and the number of CB observed in each subpopulation was averaged according to the number of nuclei (supplemental Table S3). Student's t test was performed with Excel T.TEST function (Microsoft, Redmond, WA). HeLa cells were transfected with the indicated GFP-tagged constructs and incubated 48 h on coverslips at 37 °C. Cultured cells were stained with 1 μg/ml of Hoechst 33342 dye (Fisher Scientific) for 15 min at 37 °C. Cells were washed twice with pre-warmed PBS, before incubating in DMEM with no pH indicator (GIBCO, Invitrogen). Lysotracker Red DND-99 (Molecular Probes, Eugene, OR) was used at 50 nm for 30 min. Coverslips were fixed with 4% paraformaldehyde for 15 min, then washed three times with PBS, and mounted. Lysosome volumes (red objects gated between 0.1–5 μm3) were quantified using the Volocity 6.0 measurement tool. Data from 16 stitched fields per condition were merged. ANOVA was performed with the FUNCRES.XLAM Excel add-in. Student's t test was performed with Excel T.TEST function (Microsoft). Graph and data distribution were assembled with Excel. Cells were probed with a PCM1 rabbit polyclonal antibody (A301–149A, Bethyl, Montgomery, TX) and imaged with a 60× oil-immersion objective (1.42 NA) on a Deltavision Elite DV imaging system equipped with sCMOS 2048 × 2048 pixels2 camera (GE Healthcare). Z stacks (0.2 um) were collected, deconvolved using softWoRx v5.0 (Applied Precision, Mississauga, ON, Canada) and are shown as maximum intensity projections (pixel size 0.1064 um). Cells were fixed in paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS, followed by incubation with the indicated antibodies. A Zeiss LSM 710 laser-scanning confocal microscope with a 63 × 1.4 NA oil immersion objective was used to acquire fluorescence images of cells. Z-stacks series were collected for measuring cell and peroxisome volume. Volocity 5.0 software (Perkin Elmer, Waltham, MA) was used to quantify cell volume, peroxisome number (peroxisome density) and peroxisome volume. ImageJ (NIH, Bethesda) was used to adjust brightness and contrast of images. Vero cell lysates in Laemmli buffer were analyzed by SDS-PAGE and subjected to Western blotting using: rabbit monoclonal anti-PMP70 (Abcam, Cambridge, UK), rabbit polyclonal anti-PEX14 (Millipore, Burlington, MA), mouse monoclonal anti-ATP5A (Abcam), mouse monoclonal anti-GAPDH-HRP (Novusbio, Littleton, CO), goat anti-rabbit IgG-HRP (Santa Cruz, Dallas, TX), goat anti-mouse IgG-HRP (Cedarlane, Burlington, ON, Canada). FlagBirA*-tagged protein extression was assayed by SDS-PAGE and Western blotting using: anti-FLAG M2, and biotinylated substrates visualized with Streptactin-HRP (BioRad, Hercules, CA, 1:10000 in PBS 0.1% Tween, 5% BSA). Co-IP was performed on 293 T-REx cells expressing FlagBirA*-NS3, as described in (4Duffy M.R. Chen T.H. Hancock W.T. Powers A.M. Kool J.L. Lanciotti R.S. Pretrick M. Marfel M. Holzbauer S. Dubray C. Guillaumot L. Griggs A. Bel M. Lambert A.J. Laven J. Kosoy O. Panella A. Biggerstaff B.J. Fischer M. Hayes E.B. Zika virus outbreak on Yap Island, Federated States of Micronesia.N. Engl. J. Med. 2009; 360: 2536-2543Crossref PubMed Scopus (2158) Google Scholar) and proteins analyzed by SDS-PAGE and Western blotting using anti-CEP85 antibody (Abnova, Taipei City, Taiwan H00064793-B01P, 1:500). The ZIKV genome encodes a single large polyprotein, which is cleaved by viral and host cell proteases to generate ten distinct polypeptides (Fig. 1A). To better understand ZIKV-host interactions, the ten ZIKV proteins were fused in-frame with an N- or C-terminal Flag-BirA R118G tag (constructs based on ZIKV H/PF/2013 ANO46305.1 (28Baronti C. Piorkowski G. Charrel R.N. Boubis L. Leparc-Goffart I. de Lamballerie X. Complete coding sequence of zika virus from a French polynesia outbreak in 2013.Genome Announc. 2014; 2: e00500-e00514Crossref PubMed Scopus (189) Google Scholar); individual proteins based on cleavage sites reported in (18Kuno G. Chang G.J. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.Arch. Virol. 2007; 152: 687-696Crossref PubMed Scopus (378) Google Scholar)) and expressed in HEK 293 cells, a system that displays characteristics of immature neurons (29Shaw G. Morse S. Ararat M. Graham F.L. Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells.Faseb J. 2002; 16: 869-871Crossref PubMed Scopus (575) Google Scholar) and which can be infected with ZIKV (30Monel B. Compton A.A. Bruel T. Amraoui S. Burlaud-Gaillard J. Roy N. Guivel-Benhassine F. Porrot F. Genin P. Meertens L. Sinigaglia L. Jouvenet N. Weil R. Casartelli N. Demangel C. Simon-Loriere E. Moris A. Roingeard P. Amara A. Schwartz O. Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells.EMBO J. 2017; 36: 1653-1668Crossref PubMed Scopus (85) Google Scholar) (supplemental Fig. S1A, S1B). Bait protein localization was queried using anti-Flag immunofluorescence (IF) confocal microscopy (supplemental Fig. S1C; additional IF available at ProHits-web.ca). Localizations were consistent with several previous reports on ZIKV and/or related Flaviviral proteins (supplemental Fig. S1C). Colocalization of otherwise identical N- and C-terminal-tagged ZIKV proteins indicated that the tagging moieties did not significantly affect intracellular localization (supplemental Fig. S1D). Using these cells, the virus-host protein interaction landscape was characterized using immunoprecipitation coupled with mass
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