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An Orthogonal Proteomic Survey Uncovers Novel Zika Virus Host Factors Pietro Scaturro1,2*, Alexey Stukalov1,2, Darya A

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An Orthogonal Proteomic Survey Uncovers Novel Zika Virus Host Factors Pietro Scaturro1,2*, Alexey Stukalov1,2, Darya A LETTER https://doi.org/10.1038/s41586-018-0484-5 An orthogonal proteomic survey uncovers novel Zika virus host factors Pietro Scaturro1,2*, Alexey Stukalov1,2, Darya A. Haas1, Mirko Cortese3, Kalina Draganova4,5, Anna Płaszczyca3, Ralf Bartenschlager3,6, Magdalena Götz4,5,7 & Andreas Pichlmair1,2,8* Zika virus (ZIKV) has recently emerged as a global health concern mutated in Alazami syndrome)4, LYAR (important for maintenance of owing to its widespread diffusion and its association with severe embryonic stem cell identity)5 and NGDN (a neuronal development neurological symptoms and microcephaly in newborns1. However, factor)6 (Extended Data Fig. 3a). NS4B has recently been implicated the molecular mechanisms that are responsible for the pathogenicity in the inhibition of neuronal development7. Compared to HCV-NS4B, of ZIKV remain largely unknown. Here we use human neural ZIKV-NS4B showed specific enrichment in cellular proteins associated progenitor cells and the neuronal cell line SK-N-BE2 in an integrated with the whole spectrum of ZIKV-associated pathogenesis, ranging proteomics approach to characterize the cellular responses to viral from neurodegenerative disorders and retinal degeneration (CLN6 infection at the proteome and phosphoproteome level, and use and BSG)8,9 to regulators of neuronal differentiation (CEND1 and affinity proteomics to identify cellular targets of ZIKV proteins. RBFOX2)10,11 and axonal dysfunction (CHP1 and TMEM41b)12 (Fig. 1 Using this approach, we identify 386 ZIKV-interacting proteins, and Extended Data Figs. 2–4a). Co-immunoprecipitation followed by ZIKV-specific and pan-flaviviral activities as well as host factors western blotting of transduced or ZIKV-infected cells verified that these with known functions in neuronal development, retinal defects and proteins specifically associate with arthropod-borne NS4Bs (such as infertility. Moreover, our analysis identified 1,216 phosphorylation those of dengue virus and ZIKV) and not NS4B of other Flaviviridae, sites that are specifically up- or downregulated after ZIKV infection, such as HCV; with a subset of proteins specifically binding to ZIKV- indicating profound modulation of fundamental signalling NS4B only (TMEM41b, CEND1 and CLN6; Extended Data Fig. 4b, c). pathways such as AKT, MAPK–ERK and ATM–ATR and thereby ZIKV-NS4B precipitated particularly well with ceroid-lipofuscinosis providing mechanistic insights into the proliferation arrest elicited neuronal protein 6 (CLN6), which is associated with a lysosomal by ZIKV infection. Functionally, our integrative study identifies storage disease that causes neurodegenerative late-infantile disorders ZIKV host-dependency factors and provides a comprehensive as well as retinal defects8. In human neural progenitor cells (hNPCs), framework for a system-level understanding of ZIKV-induced CLN6 redistributed to sites enriched in NS4B (Extended Data Fig. 3d). perturbations at the levels of proteins and cellular pathways. AP–LC–MS/MS experiments using CLN6 as bait identified common ZIKV, a flavivirus which is related to dengue virus, West Nile virus binding partners between NS4B and CLN6 (Extended Data Fig. 3e). and hepatitis C virus (HCV), has a single-stranded RNA genome of pos- Furthermore, CLN6 associated specifically with mTOR and TELO2, itive polarity, encoding a polyprotein that is co- and post-translationally important regulators of signalling pathways that are disrupted by processed into three structural (capsid (C), precursor membrane (prM) ZIKV7,13. Taken together, the ZIKV interactome revealed a number of and envelope (E)) and seven non-structural proteins (NS1, NS2A, new Flavivirus host-binding partners, including cellular proteins and NS2B, NS3, NS4A, NS4B and NS5)2. To comprehensively understand signalling pathway components involved in neuronal development. how ZIKV affects neuronal cells, we performed an unbiased proteomic In patients, during early stages of development, ZIKV predominantly survey to identify cellular proteins and associated complexes that inter- infects NPCs, causing microcephaly and other neurodevelopmental act with each of the ten ZIKV proteins expressed in human SK-N-BE2 injuries14. To gain insights into ZIKV-induced changes, we used a neuroblastoma cells (Extended Data Fig. 1a). Except for NS2A, all human induced pluripotent stem cell-derived neuronal differentiation ZIKV proteins were correctly expressed and processed (Extended model15. Analysis of the global proteomic changes that occurred during Data Fig. 1b, c). Affinity purification coupled with liquid chromatog- differentiation of hNPCs into neurons revealed significant upregula- raphy and tandem mass spectrometry (AP–LC–MS/MS), followed by tion of neuronal differentiation markers such as βIII-tubulin (TUBB3), Bayesian statistical modelling, identified 386 proteins specifically asso- microtubule-associated proteins 2 and 6 (MAP2 and MAP6), neuronal ciating with ZIKV proteins, resulting in 484 high-confident interactions cell adhesion molecule 1 (NCAM1), doublecortin (DCX) and ELAV- (Fig. 1, Extended Data Fig. 2 and Supplementary Table 1). We identi- like RNA binding protein 3 (ELAVL3) (Fig. 2a, Extended Data Fig. 5 fied previously reported proteins that showed bona fide interactions and Supplementary Table 2). Notably, ectopic expression of ZIKV- with flavivirus proteins, including subunits of the ATPase (ATP1A1, NS4B during differentiation specifically downregulated the expression ATP1A2, ATP1A3, ATP1B and ATP6V1H), voltage-dependent of a subset of proteins involved in neuronal differentiation (for example, anion-selective channel proteins (VDAC1, VDAC2 and VDAC3) as MAP2, MAP6, DPYSL3, DPYSL5 and CNTN2) as well as proteins asso- well as components of the cytochrome c oxidase complex (COX15, ciated with neurological diseases (for example, DOK3 and SUMO2), MT-CO2 and NDUFA4), confirming these processes as important suggesting disruption of specific developmental programs (Fig. 2b, c, targets of diverse flaviviruses3 (Fig. 1 and Extended Data Fig. 2). Extended Data Fig. 5c–e and Supplementary Tables 2, 3). Similar effects Notably, this analysis uncovered proteins linked to neurological could be seen in proteomic analysis of hNPCs infected with ZIKV in the diseases or development, particularly among the proteins that specifically presence or absence of differentiation stimuli (Extended Data Fig. 6a). interacted with capsid and NS4B (Fig. 1). For instance, the capsid- ZIKV infection led to a robust upregulation of type-I interferon- interacting proteins included LARP7 (involved in telomere stability and stimulated genes (for example, STAT1, MX1, OAS3 and IFIT1) 1Max-Planck Institute of Biochemistry, Innate Immunity Laboratory, Martinsried, Germany. 2Technical University of Munich, School of Medicine, Institute of Virology, Munich, Germany. 3Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany. 4Institute of Stem Cell Research, Helmholtz Center Munich, Neuherberg, Germany. 5Physiological Genomics, Biomedical Center, Ludwig-Maximilians-Universitaet, Munich, Germany. 6German Center for Infection Research (DZIF), Heidelberg partner site, Heidelberg, Germany. 7Synergy, Excellence Cluster for Systems Neurology, Biomedical Center, Ludwig-Maximilians-Universitaet, Munich, Germany. 8German Center for Infection Research (DZIF), Munich partner site, Munich, Germany. *e-mail: [email protected]; [email protected] 13 SEPTEMBER 2018 | VOL 561 | NATURE | 253 © 2018 Springer Nature Limited. All rights reserved. RESEARCH LETTER PARP2 PDAP1 NS1 HMBOX1 ESYT1 NS4A CEP250 E CHD8 ADAMTS12 NS5 Mitochondrial CLGN DHCR7 RPN2 ribosomal proteins ATM FAR1 NOA1 SEP15 TECR SC5D PGRMC1 MRPL42 MRPS23 MRPL2 CEP63 DMD TMEM33 MRPS22 MRPL35 PDS5A KRTCAP2 MRPL18 MRPL41 ALG8 GPRASP1 MRPS35 MRPS10 LPGAT1 MRPL21 MRPL13 MRPL24 TMX3 DDOST AAR2 Viral bait MRPS16 SCD5 MRPS34 MRPS5 MRPS25 MRPL17 PDE2A MRPL22 CCDC47 EI24 Q59GX9 MRPS7 MRPL45 ELOVL1 MRPL16 MRPL47 ERLIN1 MRPL1 CHP1 MRPS33 MRPL9 FADS2 XXYLT1 CLN6 NAP1L1 MRPS21 MRPS6 MRPS31 ICMT Prey protein KIAA0020 MRPL55 ERLIN2 MRPS11 VGF prM CEND1 MRPL3 MRPL19 AGPAT6 MRPS2 MRPS30 MRPL23 RPN1 B4DLN1 MRPL54 DERL2 Neurological MRPL10 TMEM41B B2RD90 MRPL39 MRPS14 MSH6 SV2A MRPL38 MRPL11 STT3A MRPS15 DAP3 SCD BSG disorders/ RCN2 MRPS9 MRPS27 ATP2A2 HSD17B12 MRPL37 MRPL28 neurogenesis AIFM1 MRPS26 CANX SPTLC1 HAX1 STOML2 ZC3HAV1 CALM1 AUP1 HADHB HADHA SSR3 DNAJC13 ICT1 SLC25A3 Localization F5H0B0 PEX19 LBR NS2B3 SEC61B LARP7 PTCD3 PHB GTPBP4 RAB18 EBP C7orf50 NOL6 SLC25A11 GNAI1 Cytosol DDX10 CMSS1 TUBB4A HACD3 SEC61A1 SRPK1 DIMT1 GRWD1 ZNF622 PHB2 VDAC1 DDX3Y SLC25A1 TUBA1A Plasma PPM1G DHX57 PGRMC2 VDAC2 ATP2B1 ATP1A3 SLC39A9 DNAJA1 MTDH SURF4 membrane FXYD6 NGDN BRIX1 VPS16 NAT10 CAMK2G IPO4 TSPO PPAN CEBPZ GNB2L1 HRAS NUP93 VDAC3 FAM134C SLC25A13 Nucleus MYBBP1A TIMM50 ABCB6 RFC1 DDX18 H1FX SQSTM1 NS4B RSL1D1 Capsid MTCH2 ZFR NUP205 SLC10A4 HLA-A DDX24 SET H2AFY SLC25A22 TM9SF2 Mitochondrion DKC1 POP1 TIMMDC1 TOP1 RBFOX2 CCDC137 RPSA SLC25A6 SLC38A1 LLPH RBM34 PRKDC ATP1B1 Endoplasmic NUP107 CHCHD3 SLC7A5 RRP12 RBM28 YIPF5 reticulum MAGED1 SMN1 SMPD4 SCAMP3 NIFK DDX21 KPNB1 IPO7 Golgi KRR1 GTF3C2 NUP85 LPHN2 CHPT1 BMS1 GNL3 EXOSC4 SLC3A2 YIF1A CDS2 apparatus ZCCHC8 VAPB MPDU1 DDB1 EXOSC1 FAU BOP1 NOP56 IPO9 EXOSC10 TIMM23 DHCR24 MT-CO2 EBNA1BP2 FTSJ3 EXOSC9 DDX20 TMEM165 Cytoskeleton HP1BP3 KRI1 XPOT LPCAT1 PRAF2 LYAR NUP160 TMCO1 RAB39A Mixed RPLP0 RPS15A SDAD1 RPL21 RPL5 SFXN3 NOL10 MRTO4 RPL34 RPS7 RPL11 NUP133 USMG5 ATP1A1 localization LARP1 NUP43
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