Figure S1.Affinity Peptide (One-Strep-Tag) Insertion in NS5A

Supplementary Figure Legends

Figure S1.Affinity peptide (One-STrEP-tag) insertion in NS5A.

(A) Insertion of the affinity peptide (One-STrEP-tag) sequence within the NS5A gene. An affinity peptide, termed One-STrEP-tag, which structurally resembles biotin and binds to recombinant streptavidin (Strep-Tactin), was introduced in the subgenomic replicon RNA (pSGR-JFH1 and pSGR-Luc-JFH1) in the NS5A gene. G418-resistant clone stably expressing subgenomic replicon was isolated by selective culture (300 µg/mL). Cellular lysates were used for affinity purification to isolate viral RNP complexes. SGR-Luc-JFH1-5A1ST denotes subgenomic replicon containing the luciferase gene with the One-STrEP tag insertion.

(B) Replication of One-STrEP-tagged subgenomic replicon RNA. Insertion of One-STrEP tag sequence does not interfere with the replication of subgenomic RNA. SGR-Luc-JFH1/GND represents GND mutation in the NS5B gene, which replaces active site amino acids “GDD” of NS5B with “GND”. SGR-Luc-JFH1 represents the wild type replicon RNA without the tag.

(C)Colony formation of stably transformed replicon cells. Huh 7 cells were transfected with in vitro transcribed subgenomic RNAs. After 3 weeks of selective culturing in the presence of G418 (300 µg/mL), resistant colonies were fixed with buffered 3% formaldehyde and stained with crystal violet for the visualization.

Figure S2. The Isolation of HCV viral protein complexes by affinity column chromatography from SGR-JFH1-5A1ST cells.

The affinity-column purification procedure wasperformed for two different replicon cell lines, wild type SGR-JFH1 as negative control (left) and SGR-JFH1-5A1ST as actual isolation of viral protein complex and associating host factors (right). The details are described in ‘Materials and Methods’. (A) All protein fractions from column chromatography were resolved by SDS-PAGE (10%) and visualized by silver staining. (B) Western blotting analysis of RNP complexes. This analysis shows co-fractionation of NS5A and NS3 proteins by affinity-column purification procedure in the right panel B. The identical samples from control lysates are shown in the left panel. (C)Western blot analysis of affinity-purified RNP complexes. The cellular factors chosen were identified by “MudPIT” proteomics. Following antibodies were used; ApoB; Apolipoprotein B-100, OSBP; oxysterol-binding protein.ADRP; adipose differentiation related protein; FAS; fatty acid synthase. Panels A and B represent preparation from 0.25 ml bed volume column. Panel C represents the preparation from 1.0 ml bed volume column. The peak fractions of isolated protein complexes is according to the volume/size of column bed.

Figure S3.NS5A binds OSBP and VAP-A.

(A) Full-length HCV replicon cells were transfected with pFLAG-OSBP, pFLAG-VAPA or empty vector (pFLAG-CMV) respectively. The lysates were immunoprecipitated using anti-FLAG monoclonal antibody followed by immunoblotting using anti-NS5A antibody (top panel). The lower 3 panels represent Western blot analysis of lysates derived from transfected cells of respective expression vectors. Anti-FLAG antibody detected both OSBP and VAP-A. (B) NS5A interacts with OSBP derived from genotype 1b (pEF-NS5A-1b) or JFH1, genotype 2a (pEF-NS5A-2a). Huh7 cells were co-transfected with respective Myc-tagged NS5A expression vectors and pFLAG-OSBP expression vector. Lysates were immunoprecipitated with anti-FLAG antibodies followed by immuoblotting using anti-Myc antibody (top panel). Lysates are immunoblotted with anti-Myc (middle panel, detects NS5A), or anti-FLAG antibody (lower panel, detects OSBP).

Figure S4. The subcellular distribution of NS5A, OSBP and VAP-A

(A) Subcellular distribution of NS5A, OSBP and VAP-A. HCV (JFH1) infected Huh7 cells were transfected with pFLAG-OSBP or pFLAG-VAP-A as indicated. Fourteen hours after transfection, cells grown on coverslips were fixed and immunostained using anti-FLAG and anti-NS5A antibodies. Immunofluorescence microscopy was performed as described in Materials and Methods.(B)NS5A translocates to the Golgi in response to 25-HC stimulation.Huh7 cells were transfected with an NS5A expression vector, pEF1-NS5A-MycHis-JFH1. At 18h post-transfection, the transfected cells were treated with fresh medium containing 10 M of 25-HC and incubated for another 9h prior to immunofluoescence microscopy.