1. Volume 21, Issue 13, Pages 3900-3913 (December 2017)
A Global Interactome Map of the Dengue Virus NS1 Identifies Virus Restriction and Dependency Host Factors 
Mohamed Lamine Hafirassou, Laurent Meertens, Claudia Umaña-Diaz, Athena Labeau, Ophelie Dejarnac, Lucie Bonnet-Madin, Beate M. Kümmerer, Constance Delaugerre, Philippe Roingeard, Pierre-Olivier Vidalain, Ali Amara 
Cell Reports 
Volume 21, Issue 13, Pages (December 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 3900-3913DOI: (10.1016/j.celrep.2017.11.094)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Generation of a FLAG-HA-Tagged NS1 Replicon and Purification of the DENV Replication Complex
(A) Schematic representation of the FLAG-HA (FH)-tagged NS1 replicon. Z, zeocin resistance.
(B) HeLa cell extracts were resolved on SDS-PAGE and analyzed by WB using indicated antibodies.
(C) Total cellular RNAs were extracted from WT and FH-NS1 replicon cells and relative viral RNA levels were determined by real-time qPCR. Data shown are means ± SD and are representative of two independent experiments.
(D) HeLa cells expressing WT and FH-NS1 replicon were fixed and analyzed by ultra-thin section EM. Virus-induced vesicles (Ve) and occasionally membrane tubes (T) are found around convoluted membranes (CM) closely associated to endoplasmic reticulum (ER). Insets correspond to a magnification of enlarged Ve packed in the ER lumen (white squares). Scale bar, 0.5 μm in the WT and F-HA replicon panels; 0.2 μm in the parental panel.
(E) Experimental scheme of the two-step affinity purification approach (left). WT and FH-NS1 replicon expressing cells were lysed and extracts were sequentially purified with anti-FLAG- and anti-HA-coated beads. Proteins were resolved by SDS-PAGE and visualized by silver staining (right).
(F) Schematic representation of peptides distribution (black bars) along the DENV2 polyprotein sequence (top). Number of peptides from the DENV NS proteins immunoprecipitated in Raji, HeLa, and HAP1 cell lines (bottom).
(G) FLAG-IPs from samples shown in (B) were subjected to glycerol-gradient sedimentation. Odd-numbered fractions were run on SDS-PAGE and probed with indicated antibodies.
See also Figure S1.
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4. Figure 2 A Global Map of the DENV NS1 Interactome
(A) Venn diagram highlighting 270 interactors identified by MS shared by the three cell lines.
(B) Histogram indicating statistical enrichment for specific biological processes (BP) and cellular components (CC), determined by Gene Ontology (GO) analysis.
(C) Interaction network of NS1-associated proteins identified by MS in the three replicon cell lines. Proteins were clustered into functional modules using enriched GO terms as a guideline and manual mining of literature.
See also Figure S2 and Tables S1 and S2.
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5. Figure 3
siRNA Screen Identifies DENV Host Factors that Impact DENV Infection
(A and B) Host restriction factors (HRFs) (A) and host dependency factors (HDFs) (B) identified in our RNAi screen. Data shown are representative of three independent experiments. siRNA pools targeting IFIT1 and ATP6V1B2 were included in the screen and serve as internal control for HRFs and HDFs, respectively.
(C and D) Interaction networks and functional clustering of the HRFs and HDFs whose gene silencing increased (C) or decreased (D) the percent of 2H2-positive cell by more than 2-fold, respectively. Diamonds correspond to host factors interacting with the FH-NS1 replicon in only two of the three analyzed cell lines but that physically interact with HRFs or HDFs according to literature. Small circles correspond to apparent cytotoxicity of some siRNAs as determined by fluorescence-activated cell sorting (FACS) analysis.
See also Figure S3 and Table S3.
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6. Figure 4
RACK1 and the CCT and OST Complexes Are Required for DENV Replication
(A) HeLa cells were transfected with siRNA pool targeting a representative panel of HDFs for 2 days and then challenged with DENV (MOI 2). At 48 hr post infection (hpi), level of infection and virus titer were determined and normalized to non-targeting siRNA transfected cells. Data shown are means ± SD of three independent experiments.
(B) DENV2-replicon cells were reverse transfected by the indicated siRNA pool. At 72 hr post transfection (hpt), GFP signal reflecting DENV RNA replication was quantified by flow cytometry. Data shown are means ± SD of three independent experiments.
(C) FLAG-IPs from samples shown in Figure 1B were subjected to glycerol-gradient sedimentation. Eleven-numbered fractions were run on SDS-PAGE and probed with indicated antibodies. Data shown are representative of two independent experiments.
(D and E) HeLa cells were transfected with the indicated siRNA pool and then infected with the DENV Rluc reporter virus (DV-R2A). At 4 and 24 hpi, Rluc activity reflecting DENV RNA translation (D) or replication (E) was measured. Data shown are means ± SD of two independent experiments.
(F) HeLa cells were transfected with indicated siRNA pool. At 72 hpt, cells were transfected with RNA reporter constructs. Firefly luciferase activity reflecting RNA translation was evaluated after 4 hr. (5′D-Luc-3′D): Luc flanked by DENV 5′ and 3′ UTR; (5′βg-Luc-3′A60): Luc flanked by beta globin 5′UTR and a vector sequence plus a 60-mer poly(A) tail. Data shown are means ± SD of three independent experiments.
(G) CHME and A549 cells were transfected with indicated siRNA pool and challenged 48 hr later with DENV at MOI 5. Infection was assessed 24 hpi by flow cytometry. Data shown are means ± SD of three independent experiments. Significance was calculated using a one-way ANOVA statistical test with a Dunnett’s multiple comparison test. (n.s., not significant; ∗p < 0.01; ∗∗p < 0.001; ∗∗∗p < ).
(H) HeLa cells were transfected with indicated siRNA pool, and challenged 48 hr later with DENV (MOI 5), ZIKV HD78 (MOI 2), WNV (MOI 0.4), HSV (MOI 0.3), and VSVpp (MOI 10). Infection was assessed 24 hpi by flow cytometry. Data shown are means ± SD of three independent experiments. Significance was calculated using a one-way ANOVA statistical test with a Dunnett’s multiple comparison test. n.s., not significant; ∗p < 0.01; ∗∗p < 0.001; ∗∗∗p <
See also Figure S4.
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7. Figure 5
OST N-Glycosylation Function Is Essential for DENV Replication
Silencing of different OST subunits affects DENV and ZIKV NS1 glycosylation.
(A) HEK293T cells were transfected with indicated siRNA pool. 72 h later, cells are transfected with DENV FLAG-NS1 or FLAG-NS3 plasmids. Tun, tunicamycin. Data shown are representative of three independent experiments.
(B) Same experiment as in (A) with ZIKV FLAG-NS1 plasmid. Data shown are representative of three independent experiments.
(C) HEK293T cells were transfected with the indicated FLAG-NS plasmids are incubated with increasing concentration of NGI-1. Data shown are representative of three independent experiments.
(D) Supernatants from samples shown in (C) were subjected to ELISA to quantify secreted NS1. Data shown are representative of three independent experiments.
(E) HEK293T cells were transfected with plasmids encoding FLAG-NS1 wild-type or glycosylation mutants. Data shown are representative of three independent experiments.
(F) HEK293T cells were transfected with WT NS1 or N130Q/N207Q mutant and incubated with the proteasome inhibitor MG132 (1 μM) for 16 h. Relative mutant N130Q/N207Q expression levels are compared to likewise treated WT NS1 (100%). Data shown are representative of three independent experiments.
(G) HeLa cells were pre-incubated for 24 h with increased concentration of NGI-1 or for MPA prior infection with DENV (MOI 5). MPA, mycophenolic acid.
(H) Replication kinetics of DENV2 in HeLa replicon cells challenged with NGI-1 (2 μM) or MPA (10 μM) along the experiment duration.
(I) HeLa cells were incubated for 24 h with NGI-1 and challenged in continuous presence of the drug with DENV1 (MOI 1), DENV (MOI 5), DENV2 JAM (MOI 5), DENV3 THAI (MOI 5), WNV (MOI 1), ZIKV (MOI 1), CHIKV (MOI 5), or HSV (MOI 0.3). The levels of infected cells were assessed 24 hr post infection by flow cytometry using respective anti-viral antibodies.
(J) Similarly treated primary skin fibroblasts and monocyte-derived dendritic cells (MDDC) were challenged with DENV (MOI 5) and ZIKV HD78788 (MOI 2). The levels of infected cells were assessed 24 hr post infection by flow cytometry using respective anti-viral antibodies. (G–J) Data shown are means ± SD of two independent experiments. Significance was calculated using a one-way ANOVA statistical test with a Dunnett’s multiple (D, G, and H) or Tukey comparison test (I and J). n.s., not significant; ∗∗p < 0.001; ∗∗∗p <
See also Figure S5.
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