HCV NS5A binds to active forms of Rab18.

<p>(<b>A</b>) 293T cells were co-transfected with expression plasmids encoding NS5A(SF) with tandem Strep and FLAG tags (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003513#ppat-1003513-g001" target="_blank">Figure 1A</a>) and either GFP, GFP-Rab18, GFP-Rab18(S22N), or GFP-Rab18 (Q67L). NS5A(SF) was pulled down from cell lysates with Streptactin-Sepharose at 48 hr post-transfection. Cell lysates (right panels) and eluted proteins (left panels) were subjected to Western blotting for NS5A, GFP, and β-actin. <b>B.</b> Quantitation of bound GFP-Rab18 was performed by quantitation of chemiluminescent signals on immunoblots. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003513#s2" target="_blank">Results</a> are normalized to wild-type GFP-Rab18 and represent means ± SD of four independent experiments. <b>C.</b> Distribution of NS5A and GFP-Rab18 (wild-type, S22N, or Q67L) in stable cell lines infected with JFH-1. Cells were immunostained for GFP (green) and NS5A (red) with counterstaining for lipid droplets (HCS LipidTox Deep Red, false-colored white) and DNA (DAPI, blue). Bar, 10 µm.</p

Host proteins already known to interact with NS5A and/or the HCV life cycle identified by SILAC and GeLC-MS.

<p>Proteins that specifically bind to NS5A(SF) will have incorporated heavy amino acids and thus will have a high heavy∶light (H/L) peptide ratio. PEP (Posterior error probability) refers to the probability that a peptide-spectrum match is incorrect. Intensity refers to total summed peptide intensity for the identified protein group.</p

Rab18 silencing inhibits HCV replication.

<p><b>A.</b> OR6 cells harboring a full-length genotype 1b replicon with a <i>Renilla</i> luciferase reporter were transduced with lentiviral shRNA vectors encoding a negative control nontargeting shRNA (NTshRNA), a positive control shRNA targeting PI4KA (shPI4KA), or two independent shRNAs targeting Rab18 (shRab18-A and -B). HCV replication was assessed by quantitation of <i>Renilla</i> luciferase activity (black bars) and cell viability was assessed by cellular ATP content (white bars). All values are normalized to OR6 cells transduced with NTshRNA and represent means ± SD of three independent experiments. <b>B.</b> Immunoblotting of the OR6 cells shown in panel A was performed for Rab18 and β-actin to confirm silencing of Rab18. <b>C.</b> Jc1/Gluc2A encoding a <i>Gaussia</i> luciferase reporter was used to infect Huh7.5.1 cells stably expressing NTshRNA, shRab18-A, or shRab18-B at an MOI of 1. 96 hr post-infection, <i>Gaussia</i> luciferase activity (black bars) and cellular ATP levels (white bars) were measured. All values are normalized to cells stably expressing NTshRNA and represent means ± SD of three independent experiments. <b>D.</b> Immunoblotting of the Huh7.5.1 cells shown in panel C was performed for Rab18 and β-actin to confirm silencing of Rab18. <b>E.</b> OR6 cells stably expressing GFP (first two bars), GFP-Rab18-mutA (resistant to silencing by shRab18-A; third bar), or GFP-Rab18-mutB (resistant to shRab18-B; fourth bar) were transduced with lentiviral shRNA vectors encoding a nontargeting shRNA (first bar) or shRab18-A (bars 2–4). HCV replication was assessed by quantitation of <i>Renilla</i> luciferase activity. All values are normalized to OR6 cells transduced with NTshRNA and represent means ± SD of three independent experiments. <b>F.</b> Immunoblotting of the OR6 cells shown in panel E was performed to confirm endogenous Rab18 knockdown and expression of GFP-Rab18 constructs.</p

Rab18 silencing reduces the production of infectious HCV particles.

<p><b>A.</b> Schematic of viral assembly/secretion assay. <b>B.</b> Huh 7.5.1 cell lines stably expressing shRab18-A, -B, or NTshRNA were infected with Jc1/Gluc2A at an MOI of 1. Cell culture medium was collected at day 2 postinfection, then used to infect naïve Huh7.5.1 cells. <i>Gaussia</i> luciferase activity was measured at 72 hr post-infection. These values were normalized to Huh7.5.1 cells infected with supernatant from NTshRNA-expressing cells. Values represent means ± SD of three independent experiments. <b>C.</b> Huh7.5.1 cells lines stably expressing shRab18-A, -B, or NTshRNA were transfected with <i>in vitro</i> transcribed Jc1/Gluc2A RNA. Relative quantitation of infectious particle release was performed as described above. <b>D.</b> Effect of Rab18 silencing on wild type JFH-1 secretion. Stable shRNA-expressing cell lines were infected with the JFH-1 strain of HCV at an MOI of 3. Five days post-infection, the secreted virus titer was determined using a focus-forming assay in naive Huh7.5.1 cells. Values represent means ± SD of three independent experiments. <b>E.</b> Huh 7.5.1 cell lines stably expressing shRab18-A, -B, or NTshRNA were infected with Jc1/Gluc2A at an MOI of 1. At 72 hr post-infection, the cell culture supernatant was collected for quantitation of released extracellular virus as described above. The infected cell monolayer was washed, trypsinized, and then the pellet was subjected to three rounds of freeze-thawing to release intracellular virus particles, which were then quantitated by infection of naïve Huh7.5.1 cells as described for extracellular infectious virus.</p

Identification of NS5A-binding host proteins using a proteomic strategy.

<p><b>A.</b> Diagram of Jc1(SF) showing location of FLAG and tandem Strep-Tags in domain III of NS5A. <b>B.</b> Detection of proteins specifically associated with NS5A(SF). Huh7.5.1 cells were infected with Jc1(SF) (left lane) and untagged wild-type Jc1 (right lane). Cell lysates were incubated with Streptactin-Sepharose and the affinity matrix was washed extensively followed by elution with biotin. Eluted proteins were separated by SDS-PAGE and visualized by colloidal Coomassie Blue staining. The position of NS5A(SF) is indicated by the gray arrowhead. Proteins that specifically associate with NS5A(SF) are indicated by black arrows. <b>C.</b> Schematic showing strategy of affinity purification combined with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC). Huh 7.5.1 cells were metabolically labeled with medium containing normal “light” arginine and lysine amino acids (LAA) or “heavy” L-arg-¹³C₆, ¹⁵N₄ and L-lys-¹³C₆, ¹⁵N₂ (HAA) for 6 days before infecting LAA-labeled cells with Jc1 and HAA-labeled cells with Jc1(SF) virus. Cells were harvested at day 6 post infection. Equal amounts of protein from HAA and LAA-labeled lysates were mixed and then subjected to Streptactin affinity purification as described above. <b>D.</b> Lysates from LAA-labeled, Jc1-infected cells and HAA-labeled, Jc1(SF)-infected cells were mixed and subjected to Streptactin affinity purification. The entire eluate was concentrated by precipitation, separated by SDS-PAGE and stained with Coomassie Blue prior to gel slice excision for mass spectrometry. The expected position of Rab18 is indicated with an arrow, although no discrete band is visible on this gel by Coomassie Blue staining.</p

Late endosomal DRMs are sensitive to cholesterol affecting reagents.

<p>Late endosomes were prepared from BHK cells using a sucrose step gradient, treated or not with either filipin (1μg/ml for 1 h at 37°C) or saponin (0.4% for 1 h at 4°C) and then submitted to solubilization in 1% Triton X-100 at 4°C. The lysat was submitted to an OptiPrep flotation gradient and 6 fractions of 400 µl were collected. Each fraction was analyzed by SDS-PAGE followed by an aerolysin overlay to identify the GPI-anchored proteins of BKH cells: N-CAM-120, semaphorin-7 (Sema-7), CD14 and Thy-1.</p

SNX12 silencing has no effect on VSV infection and EGFR transport and degradation.

<p>(<b>A</b>) HeLa cells treated with siRNAs against SNX12 or mock-treated were lysed. Lysates were analyzed by SDS gel electrophoresis and western blotting using with antibodies against myc, SNX3 or actin. (<b>B-C</b>) HeLa cells were treated with SNX12 siRNAs or mock-treated, and then microtubules were depolymerized or not with 10 µM nocodazole for 2 h. VSV (1 MOI) was bound at 4°C at the cell surface and cells were then incubated for 3 h at 37°C to allow VSV infection to proceed. Cells were analyzed by immunofluorescence with antibodies against VSV-G protein. Scale bar indicates 10 µm. Experiments were quantified (<b>B</b>) and representative pictures were shown in (<b>C</b>). Under these conditions ≈50% of the control cells were infected so that changes in infection rate can be best monitored, and values are normalized to the controls. Each condition is the mean of at least three independent experiments; standard errors are indicated. (<b>D</b>) After cell surface binding, EGF-biotin coupled to streptavidin^{AlexaFluor488} was endocytosed for 10 min or 50 min at 37°C in HeLa cells treated siRNAs against SNX12. Cells were labeled with anti-EEA1 or Lamp1 antibodies and analyzed by triple channel fluorescence. (<b>E</b>) HeLa cells were treated with siRNAs against SNX12 or mock treated and then incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR or α-tubulin (a-tub)<b>.</b></p

SNX12 overexpression induces an accumulation of MVB-like structures and rescues the intralumenal vesicles that incorporate the EGF receptor.

<p>(<b>A-B</b>) HeLa cells expressing GFP-SNX12 were fixed and processed for electron microscopy. Cryosections were labeled with antibodies against GFP followed by protein A-gold 10 nm (arrows). Panel A shows a cluster of several multivesicular endosomes, each being labeled with a star, and panel B shows a high magnification view of an individual multivesicular endosome. Scale bar indicates 250 nm. (<b>C</b>) Hrs (<b>D-E</b>) or SNX3 (<b>F-G</b>) was knocked down and mRFP-SNX12 (red) was overexpressed (<b>E-G</b>) or not (<b>D-F</b>). In each condition, HeLa cells were also transfected with GFP-Rab5^Q79L (green) during the last 24 h. After cell surface binding, EGF was internalized for 15 min at 37°C. Cells were processed for immunofluorescence with anti-EGFR antibodies (blue). The relative amount of EGFR in the lumen of endosome was quantified as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038949#pone.0038949-Pons1" target="_blank">[25]</a>. Results are expressed as the percentage of the total amount of EGFR. Each condition is the mean of at least three independent experiments; standard errors are indicated and results were analyzed by paired t test (***, p<0,001). (<b>D-G</b>) Representative pictures of experiments quantified in (<b>C</b>). GFP-Rab5^Q79L enlarged endosomes in each condition showed the EGFR localization after Hrs (<b>D-E</b>) or SNX3 (<b>F-G</b>) was knocked down and mRFP-SNX12 (red) was overexpressed (<b>E-G</b>) or not (<b>D-F</b>).</p

Distribution of lipid metabolism-related proteins and raft markers in late endosomes.

<p>Late endosomes were purified from BHK cells and submitted to sub-organellar fractionation after breaking the organelle by cycles of freezing and thawing followed by sucrose density gradients. 12 fractions were collected from the top and analyzed for the presence of LBPA using an ELISA assay (A) or by SDS-PAGE followed by Western blotting for the presence NPC1, MLN64, flotillin-1 and ApoE (B). GPI-anchored proteins were detected by aerolysin overlay (B).</p

SNX12 overexpression inhibits EGFR transport and degradation without affecting retrograde and recycling transport routes.

<p>(<b>A-C</b>) After cell surface binding, EGF-biotin coupled to streptavidin^{AlexaFluor488} was endocytosed for 10 min (<b>A</b>) or 50 min (<b>B-C</b>) at 37°C in HeLa cells expressing mRFP1-SNX12. Cells were labeled with anti-EEA1 (<b>A</b>) or Lamp1 (<b>B-C</b>) antibodies and analyzed by triple channel fluorescence. (<b>D</b>) HeLa cells expressing myc-SNX12 or myc-SNX3 or mock-treated were incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR, α-tubulin (a-tub) or myc<b>.</b> (<b>E</b>) After cell surface binding, Shiga toxin B-subunit conjugated to Cy3 was internalized for 10 min or 50 min at 37°C into HeLa cells expressing GFP-SNX12. Cells were labeled with anti-transferrin receptor (TfR) or Rab6 antibodies and analyzed by triple channel fluorescence. (<b>F</b>) After cell surface binding (0 min), transferrin conjugated to AlexaFluor546 (transferrin⁵⁴⁶) was internalized for 30 min or 120 min at 37°C into control cells (upper panels) or cells expressing GFP-SNX12 (lower panels). Cells were then analyzed by fluorescence. (<b>A-C and E-F</b>) Scale bar indicates 10 µm.</p