Prenylome Profiling Reveals S-Farnesylation Is Crucial For

Prenylome proﬁling reveals S-farnesylation is crucial for membrane targeting and antiviral activity of ZAP long-isoform

Guillaume Charrona, Melody M. H. Lib, Margaret R. MacDonaldb, and Howard C. Hanga,1

aLaboratory of Chemical Biology and Microbial Pathogenesis and bLaboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065

Edited by Barbara Imperiali, Massachusetts Institute of Technology, Cambridge, MA, and approved May 15, 2013 (received for review February 7, 2013) S-prenylation is an important lipid modification that targets pro- be used to detect prenylated proteins after bioorthogonal la- teins to membranes for cell signaling and vesicle trafficking in beling with fluorescent or affinity tags (20, 21). More recently, eukaryotes. As S-prenylated proteins are often key effectors for alkynyl-isoprenoids have also been developed to enable more oncogenesis, congenital disorders, and microbial pathogenesis, ro- efficient labeling and detection of prenylated proteins in bust proteomic methods are still needed to biochemically charac- mammalian cells compared with their azide-counterparts us- terize these lipidated proteins in specific cell types and disease ing Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (22, states. Here, we report that bioorthogonal proteomics of macro- 23). The application of these lipid chemical reporters has en- phages with an improved alkyne-isoprenoid chemical reporter abled the enrichment and identification of farnesylated (20) or enables large-scale profiling of prenylated proteins, as well as geranylgeranylated protein subsets (19, 21), but the general pro- the discovery of unannotated lipidated proteins such as isoform- teomic coverage of prenylated proteins has been limited. Using an specific S-farnesylation of zinc-finger antiviral protein (ZAP). Nota- alkyne-farnesol chemical reporter and improved bioorthogonal bly, S-farnesylation was crucial for targeting the long-isoform of proteomic methods, we identified over 100 candidate isoprenoid- ZAP (ZAPL/PARP-13.1/zc3hav1) to endolysosomes and enhancing labeled proteins in macrophages, including small GTPases as well the antiviral activity of this immune effector. These studies dem- as previously unannotated S-prenylated substrates such as the onstrate the utility of isoprenoid chemical reporters for proteomic zinc-finger antiviral protein (ZAP). analysis of prenylated proteins and reveal a role for protein pre- ZAP was originally identified in a rat cDNA overexpression nylation in host defense against viral infections. screen for host factors that could significantly impair replication of Moloney murine leukemia virus (MuLV) as an N-terminal antiviral effector | bioorthogonal labeling | click chemistry | protein fused to the Zeocin resistance marker (24). This original protein prenylation | lipid chemical reporter ZAP construct consisting of the first 254 amino acids of rat ZAP (rNZAP) fused to the marker (Fig. 2A and Fig. S1) and inhibited rotein S-prenylation is a covalent isoprenoid (farnesyl or the replication of various alphaviruses (25), filoviruses (26) and IMMUNOLOGY Pgeranylgeranyl) modification on cysteine (Cys) residues at retroviruses (24, 27), but did not affect host susceptibility to other carboxyl-terminal CaaX or C(X)C motifs (Fig. 1A) (1). The lipid viruses such as vesicular stomatitis virus, poliovirus, yellow fever modification increases the hydrophobicity of proteins and enhan- virus, and herpes simplex virus type 1 (25). Additional experiments ces their affinity for cellular membranes (1). Prenylated proteins suggested that rNZAP did not interfere with MuLV entry, viral have important and diverse roles in eukaryotic biology, as exem- DNA synthesis and integration, and viral RNA production in the plified by small GTPases such as K/H/Ras in cell growth (2), Rab- nucleus, but decreased the level of posttranscriptional viral mRNA family proteins in membrane trafficking (3), and lamin isoforms in in the cytoplasm (24). Similarly, rNZAP inhibited Sindbis virus nuclear matrix homeostasis (4). Because aberrant expression or (SINV) replication by blocking postentry steps of translation and mutation of prenylated proteins like K-Ras and lamin A are major amplification of incoming viral RNA (25). rNZAP is predominantly drivers of human diseases like cancer or progeria, respectively, localized in the cytoplasm at steady state but shuttles between prenyltransferase inhibitors that interfere with lipidation of these the cytoplasm and the nucleus in a CRM1-dependent manner proteins and their function are under clinical development (5, 6). (28). rNZAP is also proposed to bind cytoplasmic viral mRNA In addition to cellular proteins, virulence factors from viruses (7- through its second and fourth CCCH-type zinc-fingers (26, 29) 10) and bacterial pathogens (11-14) can be S-prenylated by host although recent structural studies suggest a role for all four zinc- enzymes to enhance microbial infection. The analysis of S-preny- fingers in forming an RNA binding groove (30). ZAP recruits lated proteins is therefore crucial for understanding fundamental p72 DEAD-box (31) and DHX30 DEXH-box (32) RNA heli- mechanisms of cell biology and human disease, as well as the cases, and the RNA processing exosome (33) for optimal depletion characterization of drugs targeting protein prenylation. of viral mRNA. Although early ZAP studies were conducted Although bioinformatics predict hundreds of prenylated pro- with rNZAP, the analysis of full-length rat ZAP (rZAP), which teins in eukaryotes based on Cys-Aaa-Aaa-Xaa (CaaX) or Cys- bears an additional WWE domain predicted to mediate specific Xaa-Cys [C(X)C] motifs of known substrates (15), only a small protein–protein interactions in ubiquitin and ADP ribose fraction has been characterized biochemically and experimen- tally validated. Although detergent partitioning methods and radioactive lipid labeling have been useful for characterizing Author contributions: G.C., M.M.H.L., M.R.M., and H.C.H. designed research; G.C. and prenylated proteins, more sensitive methods for monitoring M.M.H.L. performed research; G.C., M.M.H.L., and M.R.M. contributed new reagents/ protein prenylation are still needed (16). To this end, chemical analytic tools; G.C., M.M.H.L., M.R.M., and H.C.H. analyzed data; and G.C. and H.C.H. reporters of prenylated proteins that provide better sensitivity than wrote the paper. traditional radioactive labeling have now been developed (17, 18). The authors declare no conflict of interest. For example, a biotin geranyl diphosphate analog can be used with This article is a PNAS Direct Submission. engineered prenyltransferases to visualize and profile geranylger- 1To whom correspondence should be addressed. E-mail: [email protected]. anylated proteins in vitro (19). Alternatively, azide-derivatives of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. farnesol or geranylgeraniol or their diphosphate analogs can 1073/pnas.1302564110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1302564110 PNAS | July 2, 2013 | vol. 110 | no. 27 | 11085–11090 Downloaded by guest on September 25, 2021 suggesting that regulation of ZAP activity can enhance or even amplify IFN responses (36). In addition, ZAPS and ZAPL are both polyADP ribosylated and have been implicated in stabi- lization of mRNA during cellular stress (37). Interestingly, ZAPS polyADP ribosylation is selectively elevated during cellular stress compared with ZAPL (37). These results highlight a potential duality in the regulation and functions of ZAP isoforms. The role of S-prenylation on ZAPL (PARP-13.1/zc3hav1) localization and antiviral function has not been investigated. Here, we show that S-prenylation enhances the membrane targeting and antiviral activity of ZAPL. Results and Discussion Proteomic Analysis of Prenylated Proteins in RAW264.7 Macrophages. To identify lipid-modiﬁed proteins involved in immune responses to microbial infections, we performed a large-scale proﬁling

Fig. 1. Visualization and identification of prenylated proteins in RAW264.7 macrophages. (A) Scheme of S-prenylated proteins with a CaaX motif (Left) and dually S-geranylgeranylated Rab proteins with a C(X)C motif (Right). (B) Metabolic labeling of cells with alk-FOH prenylation reporter and sub- sequent CuAAC ligation with bioorthogonal detection tags for imaging or proteomics. (C and D) RAW264.7 macrophages pretreated for 12 h with 10 μM lovastatin and then incubated for 12 h with 50 μM alk-FOH or DMSO as a control. In C, cell lysates were reacted with az-rho by CuAAC, and proteins were separated by SDS/PAGE for visualization by fluorescence gel scanning. Coomassie blue (CB) staining demonstrates comparable loading. In D, lysates from lovastatin-treated cells were reacted with az-azo-biotin by CuAAC for enrichment of alk-FOH–labeled proteins with streptavidin beads and identification by mass spectrometry. For each high-confidence identified protein, the difference of assigned peptide spectral counts from the alk-FOH and DMSO samples was plotted. Proteins with a carboxyl-terminal CaaX or C (X)C motif are shown in red. Several known prenylated proteins are labeled in black, and ZAP, the highest ranked protein not known to be prenylated, is labeled in bold blue.

conjugation systems (34) (Fig. 2A and Fig. S1), suggests similar antiviral activity against MuLV (24). Recent reports have suggested that human ZAP (hZAP) recruits both the 3′ and 5′ mRNA degradation machinery because it binds adenylase poly(A)-specific ribonuclease to remove the poly(A) tail and the decapping complex Dcp1a/Dcp2 to remove the cap structure (27). There are two ZAP isoforms arising from alternative splicing: ZAP-long (ZAPL) and ZAP-short (ZAPS) (Fig. S1). Both have the same amino acid sequence, but ZAPS lacks the carboxyl- Fig. 2. S-farnesylation of Cys993 excludes murine ZAPL from the cytosol. (A) terminal poly(ADP ribose) polymerase (PARP)-like domain Schematic representation of protein domains of rNZAP, rZAP, and mouse HA- – present in ZAPL (Fig. 2A) (35). Even though the PARP-like tagged ZAP constructs. (B D) 293T or MEFs were transfected with pCMV-HA- ZAPL, pCMV-HA-ZAPL-SaaX, or pCMV-HA-ZAPS (shown in A) and labeled with domain of ZAPL is predicted to be inactive, ZAPL/S isoforms 50 μM alk-FOH for 4 h. In B and C, 293T cell lysates were subjected to anti-HA have been annotated as PARP-13.1/2 respectively. Human ZAPL immunoprecipitation, reacted with az-rho by CuAAC, separated by SDS/PAGE, exhibits stronger antiviral activity than hZAPS against MuLV ex- and visualized by fluorescence gel scanning. Comparable protein loading was pression and Semliki forest virus infection (35) whereas both confirmed by anti-HA Western blotting. In C, cells were pretreated for 1 h with isoforms prevent HIV-1 infection (27). The ZAP isoforms are 10 μM prenyltransferase inhibitors (FTI-297, farnesyltransferase inhibitor; relatively broadly expressed in human tissues (35), but the mRNA GGTI-2133, geranylgeranyltransferase-I inhibitor) as indicated before alk-FOH α labeling. In D, MEFs were detergent fractionated into nuclear (N), membrane expression of hZAPS is markedly increased by IFN treatment (M) and cytosolic (C) fractions, and their purity was assessed by anti-histone (36). hZAPS is also more active than hZAPL in enhancing RIG-I- H3, anti-calnexin, and anti-GAPDH Western blotting, respectively. Cellular dependent signaling in response to 5′-triphosphate-modified RNA, localization of HA-ZAP was assessed by anti-HA Western blotting.

11086 | www.pnas.org/cgi/doi/10.1073/pnas.1302564110 Charron et al. Downloaded by guest on September 25, 2021 of prenylated proteins in the mouse macrophage line RAW264.7 expressing hemagglutinin epitope (HA)-tagged mouse ZAP (38) using the isoprenoid chemical reporter alkynyl-farnesol (alk- (HA-ZAP) followed by anti-HA immunoprecipitation, and FOH) (22, 23) and CuAAC (Fig. 1B). Alk-FOH targets the CuAAC with az-rho and in-gel fluorescence scanning, indicated substrates of all three prenyltransferases in cells: CaaX S-far- that ZAPL, but not ZAPS, is indeed prenylated (Fig. 2B and Fig. nesylated and S-geranylgeranylated proteins, as well as C(X)C S4A). Because protein S-prenylation is sometimes followed by RabGTPases (22). In-gel fluorescence profiling of RAW264.7 protein S-palmitoylation to increase membrane affinity (42), cell lysates, reacted with an azide-functionalized fluorophore, we also labeled HA-ZAPL–expressing HeLa cells with alk-16, azido-rhodamine (az-rho) (39), demonstrated that a diverse a chemical reporter of palmitoylation (39), but palmitoylation of repertoire of proteins are metabolically labeled by alk-FOH (Fig. ZAPL was not detected (Fig. S4A). We then evaluated whether 1C). Cell lysates were then reacted with a cleavable affinity tag ZAPL is prenylated at cysteine (Cys) residue 993 of the CaaX (az-azo-biotin) (40) for enrichment of alk-FOH–labeled proteins motif. Alk-FOH labeling of HEK293T and HeLa cells trans- with streptavidin beads, selective elution, and gel-based proteo- fected with a plasmid bearing a Cys-to-serine (Ser) mutation in mic identification by mass spectrometry (Fig. 1B). Coomassie HA-ZAPL, termed HA-ZAPL-SaaX (Fig. 2A), showed a signifi- blue staining of proteins retrieved with streptavidin beads and cant reduction in the level of ZAPL prenylation (Fig. 2B and Fig. sodium dithionite (Na2S2O4) elution demonstrates the specificity S4B). To assess whether ZAPL is farnesylated or geranyger- of alk-FOH and CuAAC labeling methods (Fig. S2A). Proteins anylated, HEK293T and HeLa cells expressing HA-ZAPL were identified in three independent experimental runs were compiled treated with farnesyltransferase or geranylgeranyltransferase-I and categorized into high- and medium-confidence lists on the inhibitors (FTI-297 or GGTI-2133, respectively) before alk-FOH basis of the total number of assigned spectra and the fold- labeling. Although GGTI-treated cells had the same fluorescence increase above control samples that were not labeled with alk- level as nontreated cells, FTI-treated cells showed a marked FOH. For this analysis, we selectively identified 114 proteins by decrease, suggesting that the long-isoform of ZAP (ZAPL) is alk-FOH labeling compared with control samples, with 52 and S-farnesylated at Cys993 (Fig. 2C and Fig. S4C). 62 proteins assigned to high- and medium-confidence lists, respectively (Fig. 1D and Tables S1 and S2). The analysis of sub- S-Farnesylation Controls Membrane Targeting and Cellular Localization cellular distribution suggests that 60% of high-confidence hits of ZAPL. We next investigated whether S-farnesylation of ZAPL were membrane-associated proteins whereas 21% were mito- affected membrane partitioning and cellular localization. For chondrial proteins (Fig. S2B). Of the high-confidence list, 35% these studies, mouse embryonic fibroblasts (MEFs) were (23 + 12) of hits bear a carboxyl-terminal CaaX or C(X)C motif, transfected with HA-ZAPL, HA-ZAPL-SaaX, or HA-ZAPS and respectively (Table S1). Of these putative prenylated proteins, fractionated into cytoplasmic, membrane, and nuclear fractions. 61% of high- and medium-confidence hits have been reported in Each fraction was analyzed for ZAP distribution by anti-HA previous labeling and enrichment studies with biotin, azide, or immunoblot along with histone H3, calnexin, and GAPDH as alkyne chemical reporters in other cell types (Tables S1 and S2). controls for nuclear, membrane, and cytoplasmic fractions, re- These proteins include K-Ras, Cdc42, Lamin-B1, DnaJA2, spectively (Fig. 2D). Although ZAPS was found in nuclear, Rap2C and Rab proteins (Fig. 1D and Tables S1 and S2). In membrane, and cytosolic fractions, ZAPL was markedly de- addition, RhoA, Ptp4A, Ykt6, Rac2, Brox and RhoG, which pleted from the cytosol (Fig. 2D). This cellular fractionation was have predicted prenylation sites, were recovered in our alk-FOH attributed to S-farnesylation as ZAPL-SaaX was redistributed to IMMUNOLOGY proteomic dataset (Fig. 1D and Tables S1 and S2). In comparison the cytosol similar to ZAPS (Fig. 2D). MEFs transfected with the with previous proteomic studies that targeted subsets of S-preny- HA-ZAP constructs were also analyzed by immunofluorescence lated proteins (19-21, 23), our proteomic analysis of alk-FOH–la- using confocal microscopy. Although ZAPL exhibited punctate beled proteins recovered both farnesylated and geranylgeranylated clusters, ZAPL-SaaX showed a more diffuse staining, similar to proteins, as well as many other candidate isoprenoid-modified ZAPS (Fig. 3A). Cotransfection of plasmids expressing HA-ZAP proteins (Tables S1 and S2). To validate our alk-FOH–labeled and cellular markers indicated that ZAPL localizes to lysosomes proteins in our dataset, we biochemically characterized a ca- (LAMP1-GFP) and late endosomes (GFP-Rab7), but not early nonical CaaX-containing farnesylated protein (DnaJA2) and endosomes (GFP-Rab5) (Fig. 3B). This localization was not an unpredicted substrate (Pcbp1). Analysis of GFP-tagged observed with ZAPL-SaaX or ZAPS (Figs. S5–S7). S-farnesyla- DnaJA2 constructs demonstrated that alk-FOH labeled this tion is thus required for targeting ZAPL to late endosomal and CaaX-containing protein at the predicted site of S-prenylation, lysosomal compartments. which also was uniquely sensitive to the farnesyltransferase inhibitor (FTI-297) (Fig. S3 A and B) and consistent with previous S-Farnesylation Regulates ZAPL Antiviral Activity. We then de- reports of farnesylation on this chaperone protein (20). In addi- termined whether isoform-specific S-farnesylation was crucial for tion, we demonstrate that HA-tagged Pcbp1, a protein implicated ZAPL antiviral activity using Sindbis virus (SINV) as a pro- in nucleic acid binding (41), is also labeled by alk-FOH, the ma- totypic alphavirus for these infection assays. Because murine jority of which appears to be on Cys355 at the carboxyl terminus ZAP mRNA level is up-regulated by type I IFN (43) and ZAP C D exhibits homotypic interactions (44), we used Stat1-deficient and insensitive to prenylation inhibitors (Fig. 3 and ). These − − – (Stat1 / ) MEFs that are defective in IFN signaling (45) to results demonstrate that proteomic analysis of alk-FOH labeled − − proteins enables profiling of known prenylated proteins and dis- mitigate the effects of IFN-induced endogenous ZAP. Stat1 / covery of unanticipated isoprenoid-modified proteins. Notably, we MEFs transfected with HA-ZAP constructs were thus infected identified the long-isoform of the zinc-finger antiviral protein with a SINV encoding enhanced green fluorescent protein (TE/ (ZAPL) as a high-confidence hit, which was not previously an- 5′2J/GFP) (46) at a multiplicity of infection (MOI) of 10 for 24 h notated as a prenylated protein. before immunostaining and flow cytometric analysis of the HA tag (ZAP) and EGFP (virus) expression. This analysis allowed Long-Isoform of ZAP is S-Farnesylated. S-prenylation of ZAP was direct comparison of virus infection in transfected and non- investigated further to validate our alk-FOH proteomic data and transfected cells in the same sample by gating on the cells with − − explore the impact of this lipid modification on ZAP subcellular high and low HA staining, respectively. Transfected Stat1 / localization and antiviral activity. ZAP amino acid sequence MEFs with low HA staining were infected at comparable levels analysis indicates that ZAPL, but not ZAPS, bears a carboxyl- (26%–28%) to vector control samples (32%) (Fig. 4A, lower terminal CaaX motif for protein prenylation (Fig. 2A). Alk-FOH quadrants, and Fig. 4B, gray bars). In addition, transfected cells labeling of HEK293T and HeLa cells transfected with plasmids expressed similar levels of different HA-ZAP proteins, as

Charron et al. PNAS | July 2, 2013 | vol. 110 | no. 27 | 11087 Downloaded by guest on September 25, 2021 plus strand genomic RNA (25), S-farnesylation may further enhance this antiviral activity by directing ZAPL to endocytic membranes to interact with incoming virus. Interestingly, SINV RNA replication has been observed to initiate in plasma membrane-associated spherules (47), which at later times can be in- ternalized and form cytopathic vacuoles bearing markers of both endosomal and lysosomal membranes (48). ZAPL enriched on endo/lysosomal membranes could therefore also have an inhibitory effect during SINV replication. It will be interesting to evaluate whether S-farnesylation will also enhance the antiviral activity of ZAPL against other viruses such as MuLV (35) and HIV-1 (24, 27). The results presented here suggest that S-farnesylated ZAPL exhibits a unique antiviral activity on cellular membranes, which may be important for the development of new antiviral strategies. Overall, these studies highlight how bioorthogonal proteomics of protein S-prenylation can reveal insights into host–pathogen interactions that should be useful for exploring other biological pathways and human diseases. Materials and Methods Cell Culture, Tranfections, Virus Infections, and Flow Cytometry. RAW264.7 macrophages, HEK293T, HeLa cells, wild-type and Stat1−/− MEFs were grown in DMEM with 10% (vol/vol) FBS. HEK293T cells were transfected using X- tremeGENE 9 DNA Transfection Reagent (Roche) whereas HeLa cells and MEFs were transfected using Lipofectamine 2000 (Invitrogen). Sindbis virus encoding the enhanced green fluorescent protein (EGFP) from a duplicated subgenomic promoter (TE/5′2J/GFP) has been previously reported (46). – Fig. 3. S-Farnesylation dependent clustering of ZAPL to endo/lysosomes. Stocks were prepared and titers determined on BHK-J cells with 10-fold se- (A) MEFs grown on coverslips were transfected with pCMV-HA-ZAPL, pCMV- rial dilutions of sample, and then plaques were visually enumerated after HA-ZAPL-SaaX, or pCMV-HA-ZAPS and stained with anti-HA (red) and crystal violet staining, as previously described (25); multiplicities of infection TOPRO-3 (blue). Insets are enlargements of the white-squared regions. (MOI) were calculated based on BHK-J–derived titers. For flow cytometry, μ (Scale bar: 10 m.) (B) MEFs were also cotransfected with pCMV-HA-ZAPL cells were fixed with PBS plus 3.7% paraformaldehyde and then per- and plasmids expressing cellular markers LAMP1-GFP, GFP-Rab7, or GFP- meabilized and blocked with PBS plus 0.1% Triton X-100 plus 2% FBS. Cells Rab5 (green) as indicated. (Scale bars: 10 μm.)

determined by the percentage of cells with high HA staining and the mean fluorescence intensity (MFI) of those cells (Fig. 4A, − − upper quadrants). Only 5% of HA-ZAPL expressing Stat1 / MEFs were infected whereas cells expressing the nonfarnesylated HA-ZAPL-SaaX and HA-ZAPS proteins had higher levels of infected cells (14%–15%; Student t test: P = 0.00016 and 0.00003, respectively) (Fig. 4B, black bars). ZAPL inhibited SINV to a significantly greater extent than ZAPS (Fig. 4C), with ZAPS demonstrating only 65% of the antiviral activity of ZAPL. These data are consistent with previous ZAP studies with MuLV (35). This increased antiviral activity is mostly attributed to ZAPL S- farnesylation because HA-ZAPL-SaaX expression resulted in infected cell levels similar to HA-ZAPS (Fig. 4 B and C). The S- farnesylation–dependent antiviral activity of ZAPL was also observed at a lower MOI (Fig. S8 A and B) and in HEK293T cells (Fig. S8 C–F). These results demonstrate that S-farnesylation significantly enhances the antiviral activity of ZAPL.

Concluding Remarks. Prenylation provides an essential membrane- targeting mechanism that controls the functions of many proteins in eukaryotic biology. The direct biochemical analysis of these lipidated proteins can therefore reveal important activities in Fig. 4. Antiviral activity of ZAPL is regulated by S-farnesylation. Stat1−/− cellular membranes not readily apparent by monitoring protein MEFs were transfected with pCMV-HA (vector), pCMV-HA-ZAPL, pCMV-HA- expression alone. The application of an alkyne-farnesol reporter ZAPL-SaaX, or pCMV-HA-ZAPS (shown in Fig. 2A) and infected with Sindbis and improved bioorthogonal proteomics described here has en- virus encoding the enhanced green fluorescent protein (EGFP) from a du- abled large-scale proteomic analysis of known prenylated pro- plicated subgenomic promoter (TE/5′2J/GFP) with multiplicity of infection teins, such as small GTPases, as well as unannotated substrates (MOI) of 10 for 24 h. Virus replication and ZAP protein levels were examined like ZAPL. Our discovery and characterization of ZAPL lip- by flow cytometry using GFP fluorescence and anti-HA staining, respectively. idation demonstrates that S-farnesylation enhances the mem- After gating (shown in A), nontransfected and transfected cells expressing ZAP from the same culture were analyzed for the percentage of these cells brane targeting and inhibitory activity of this antiviral protein = = – that were infected (shown in B). *P 0.00016, **P 0.00003 by Student against SINV (Figs. 2 4). As expression of ZAPS constructs has t test; error represents SD, n = 3. (C) Percentages in B were normalized such been shown to inhibit SINV infection by blocking translation of that the difference in infection rates for vector control and HA-ZAPL– incoming viral RNA and thus amplification of newly synthesized transfected cells was set at 100% antiviral activity.

11088 | www.pnas.org/cgi/doi/10.1073/pnas.1302564110 Charron et al. Downloaded by guest on September 25, 2021 were then incubated with mouse anti-HA antibody (1/1,000, H3663; Sigma), pH 7.4, 150 mM NaCl) and incubated with 300 μL of prewashed streptavidin- washed three times, and stained with goat anti-mouse antibody conjugated agarose beads (20357; Thermo Scientific) for 1 h at room temperature. The to Rhodamine Red-X (1/1,000, R6393; Invitrogen). Results were analyzed beads were washed once with PBS plus 1% SDS, thrice with PBS, and twice with FlowJo software. with ABC buffer (50 mM ammonium bicarbonate). The beads were incubated in 500 μL of ABC buffer containing 8 M urea, 10 mM TCEP, and Metabolic Labeling, Immunoprecipitations, and CuAAC. Metabolic labeling of 20 mM iodoacetamide for 0.5 h at room temperature, and then washed μ RAW264.7, HEK293T, and HeLa cells with alk-FOH (50 M, 4 h) or DMSO twice with ABC buffer. Proteins were eluted by incubating the beads twice fi control was performed in DMEM and 2% charcoal- ltered FBS. For coincu- in 250 μL of ABC buffer containing 1% SDS and 25 mM Na S O for 1 h at μ 2 2 4 bation with inhibitors, cells were pretreated for 1 h with FTI-277 (10 M) or room temperature. Proteins from the pooled supernatants were concen- GGTI-2133 (10 μM) before alk-FOH metabolic labeling. Chemical syntheses of trated using an Amicon Ultracel-10K (UFC501096; Millipore). Samples were alk-FOH (22), az-rho (39), and az-azo-biotin (40) have been previously then subjected to SDS/PAGE and staining with Coomassie blue. DMSO and reported. Alk-FOH labeled cells were lysed [4% (wt/vol) SDS, 50 mM trie- alk-FOH lanes of the gel were then cut for trypsin digestion and peptide thanolamine pH 7.4, 150 mM NaCl, EDTA-free Roche protease inhibitor extraction. Extracted peptides were dried and resuspended in 0.1% tri- mixture, 1 mM PMSF], and protein concentration was determined by the fl BCA assay (Pierce). Proteins (50 μg) were conjugated to az-rho in 50 μL with uoroacetic acid for mass spectrometry. CuAAC reactants [az-rho (0.1 mM), Tris(2-carboxyethyl)phosphine hydro- chloride (TCEP, 1 mM), Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, Cell Fractionation. Qproteome Cell Compartment Kit (Qiagen; 37502) was ’ 0.1 mM), and CuSO4·5H2O (1 mM)] for 1 h at room temperature, and methanol used following the manufacturer s procedure, and Western blots were precipitated before SDS/PAGE. For immunoprecipitations, 4% SDS was performed using mouse anti-HA (1/1,000, H3663; Sigma), rabbit anti-histone replaced by 1% Brij 97 as the detergent, and 400 μg of proteins in 250 μL H3 (1/2,000, 06–755; Millipore), rabbit anti-calnexin (1/1,500, ab22595; were rocked at 4 °C for 1 h with 15 μL of packed anti-HA agarose-antibody Abcam), or rabbit anti-GAPDH (1/5,000, ab70699; Abcam) antibodies, fol- conjugate (A2095; Sigma). The beads were washed thrice (1% Triton X-100, lowed by HRP-conjugated donkey anti-mouse (1/15,000, 715–035-150; Jack- 1% sodium deoxycholate, 0.1% SDS, 50 mM triethanolamine pH 7.4, son ImmunoResearch) or HRP-conjugated goat anti-rabbit (1/15,000, 12–348; 150 mM NaCl), and proteins were conjugated to az-rho in 50 μL of PBS with Millipore) antibodies. CuAAC reactants for 1 h at 4 °C and washed thrice again before SDS/PAGE. fl In-gel uorescence scanning was performed using a Typhoon 9400 imager Microscopy. For determination of ZAP localization, transfected HEK293T or fi (Amersham Biosciences; 532 nm laser, 580 nm lter 30BP). Western blots MEF cells were fixed with PBS containing 3.7% paraformaldehyde, per- for HA-tagged proteins were performed using a mouse anti-HA antibody meabilized with PBS containing 0.1% Triton X-100, and blocked with PBS (1/1,000, H3663; Sigma) followed by HRP-conjugated donkey anti-mouse containing 2% FBS. Cells were then incubated with anti-HA antibody (1/1,000, antibody (1/15,000, 715–035-150; Jackson ImmunoResearch). H3663; Sigma), washed thrice, and stained with goat anti-mouse antibody For proteomic experiments, RAW264.7 macrophages were pretreated with conjugated to Rhodamine Red-X (1/1,000, R6393; Invitrogen). Cells were lovastatin (10 μM, 12 h); then alk-FOH was added (50 μM, 12 h) and cells fi were harvested, lysed in 1 mL of ice-cold hypotonic buffer (5 mM trietha- incubated with TOPRO-3 (1/1,000; Invitrogen) as a nal step.

nolamine, pH 7.4, 5 mM MgCl2, EDTA-free Roche protease inhibitor mixture, 1 mM PMSF), and solubilized by dilution with 1 mL of 2× SDS buffer (8% SDS, ACKNOWLEDGMENTS. G.C. acknowledges the Weill-Cornell/Rockefeller/ 100 mM triethanolamine, pH 7.4, 300 mM NaCl) plus 2 μL of Benzonase Sloan-Kettering Tri-institutional Program in Chemical Biology. M.M.H.L. acknowledges support from the Northeast Biodefense Center. M.R.M. nuclease (E1014; Sigma). Cell lysates (20 mg) were then reacted with az- acknowledges support from National Institutes of Health (NIH) Grant azo-biotin (40) in 20 mL with CuAAC reactants (same as above) for 2 h at AI057905, the Irma T. Hirschl/Monique Weill-Caulier Trust, the Greenberg room temperature. Methanol-precipitated and washed protein pellets were Medical Research Institute, and the Starr Foundation. H.C.H. acknowl- IMMUNOLOGY resuspended in 2 mL of 1× SDS buffer plus 10 mM EDTA. Proteins (15 mg) edges support from the Ellison Medical Foundation and NIH/National were diluted with 8 mL of Brij buffer (1% Brij 97, 50 mM triethanolamine, Institute of General Medical Sciences Grant 1R01GM087544.

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