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1 Dynamic Interaction of Stress Granule, and Mediates Multiple Functions in Hepatitis C Virus Infection Véronique Pène, Qisheng Li#, Catherine Sodroski, Ching-Sheng Hsu, T. Jake Liang# Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA Running Head: Localizations and Functions in HCV infection #Address correspondence to Qisheng Li, liqisheng@niddk.nih.gov, or T. Jake Liang, jakel@bdg10.niddk.nih.gov. V.P. and Q.L. contributed equally to this work. 1

2 SUPPLEMENTAL FIGURES A Mito Mito Mito Mito Mito HCV HCV 3 UTR Ctrl Catalase Catalase Catalase LC3B LC3B LC3B HCV Catalase HCV 3 UTR Ctrl HCV HCV 3 UTR Catalase D E Mito Catalase Mito F Ctrl C HCV 3 UTR Ctrl B Catalase 2

3 Figure S1. HCV 3 UTR RNA or HCV-induced granules do not colocalize with mitochondria, peroxisomes or autophagosomes. Huh cells were transfected with EGFP control (Ctrl) or HCV 3'UTR RNA for 24 h or infected with HCVcc for 48 h., and various cellular compartment markers were stained and examined by confocal microscopy. (A) granules are not localized to mitochondria (Mito, stained by Mitotracker). (B) granules are not localized to peroxisomes (Catalase as a marker). (C, D) granules are not on peroxisomes (Catalase as a marker)(c) or autophagosomes (LC3B as a marker)(d). (E, F) Upon HCV infection, granules partially co-localize with but not with mitochondria (Mito, stained by Mitotracker)(E) or peroxisomes (Catalase as a marker)(f). Magnifications of two independent selected areas are shown in the right panels: small granules at the LD surface (orange area) and big granules not associated with (blue area). (A-D) Magnifications of selected area are shown in the right panels. (A, E, F) Colors in far right pictures were digitally changed for better visualization: (A) and (E, F), green instead of magenta. Scale bars, 20 µm. 3

4 A B DDX6 DDX6 DDX6 C D HCV _ + _ + _ + EDC4 WB: anti- HC IP: anti-igg anti- anti- EDC4 _ + WB: anti- EDC4 WCL Figure S2. HCV-induced granules co-localize with SG and P- body markers. (A-C) Huh cells were infected with JFH-1 HCVcc for 48 h, and then immunostained for, and various SG and P-body markers. (A), and co-localize with (another SG marker). (B, C), and co-localize with P-body markers DDX6 (B) and EDC4 (C). Scale bars, 20 µm. (D) Huh cells were mock infected or infected with JFH-1 HCVcc for 48 h. Immunoprecipitations (IP) were performed on whole cell lysate (WCL) with anti-, anti- or IgG control antibody, followed by Western blot using anti- or anti- antibody. HC, IgG heavy chain. 4

5 A HCV 24 h HCV 8 h HCV 4 h HCV 0.5 h HCV 48 h Mock 80% 60% h 27 HCV 72 h 100% 40% 100% 20% B h 84 80% 0% 60% 80% 40% 60% 40% 20% 20% 0% 0% h 42 Cell distribution 100% No granules granules granules at LD Jc JF Ct SG Co 1 rl / H granules n1 at LD R2 1 a Mock 30 min 4h 8h Ct rl SG 24 h R2 a JF H 48 1h Co Jc n h h8 Figure S3. Dynamic association of with HCV core protein and during HCV h4 infection. (A) Huh cells were mock infected or infected with JFH-1 HCVcc for 0.5 to 72 h., HCV core protein and were stained and their subcellular localizations and nim 03 5 kc

6 associations were examined. Magnifications of selected areas are shown in the right panels. Colors in far right panels were modified for better visualization: HCV core protein, green instead of magenta. Scale bars, 10 µm. (B) Quantification of co-localization with HCV core protein and. Five to ten confocal microscopic pictures from at least three independent experiments with a total of at least 30 cells were analyzed for co-localization signals. Percentages of cells without granular structures (light grey striped bars) or presenting structures that are either not co-localized (blue stripped bars) or co-localized with only (green striped bars) or with both HCV core protein and (green bars) are shown. 6

7 Mock HCV 72 h HCV 48 h HCV 24 h HCV 8 h HCV 4 h Figure S4. Dynamic redistribution of SGs towards during HCV infection. Huh cells were mock infected or infected with JFH-1 HCVcc for 4 to 72 h. SG proteins and and were stained and examined by confocal microscopy. Magnifications of selected areas are shown in the right panels. Colors in far right pictures were changed for better visualization: (green instead of magenta) and (red). Scale bars, 20 µm. 7

8 A B Figure S5. SGs are not induced in the absence of infectious HCV. Huh cells were inoculated for 30 min with culture supernatant of uninfected cells (A) or with heat-inactivated (10 min at 100 C) JFH-1 HCVcc (B). and were then immunostained and examined by confocal microscopy. Scale bars, 20 µm. 8

9 A mrna (relative value) 1.2 ** si B HCV RNA (relative value) 1.2 Intracellular Extracellular ** ** si C si Nuclei 0±4 3±6 D E H RLU (relative value) 1.2 ** si RLU (relative value) 1.2 ** si si si F G I Gene expression, mrna (relative value) TIA1 TIAL1 ** ** ** sitia1 si sitial1 HCV RNA (relative value) 1.2 * ** * ** si Intracellular Extracellular sitia1 ** ** sitial1 RLU (relative value) 1.2 * si Figure S6. Effects of or SG proteins silencing in cells on productive HCV infection. Huh cells were transfected with non-targeting control () or various indicated sirnas for 3 days and then infected with JFH-1 HCVcc (A-C, F-H) or transfected 9

10 with HCV IRES RNA (D) or JFH1-Rluc subgenomic replicon RNA (E, I). (A) sirna-mediated knockdown efficiency, examined by gene expression assay. (B) Quantification of HCV RNA levels in cells (intracellular) or supernatant (extracellular) by RT-qPCR in -silenced cells. (C) HCV core protein staining by immunofluorescence in -silenced cells. Nuclei (blue) were stained with DAPI to determine the total cell numbers and percentage of infected cells. The ratio of infected cells as compared to (as 0) was shown on top left corner of each image. (D) HCV IRES-mediated translation assay in -silenced cells, performed at 24 h after transfection of the IRES replicon RNA that harbours firefly luciferase reporter gene. (E) HCV subgenomic replicon assay in silenced cells. Renilla luciferase activity was measured 2 days post-tranfection to determine the level of viral RNA replication. (F) Knockdown efficiencies of various indicated sirnas in Huh cells. (G) Quantification of HCV RNA levels in cell lysate (intracellular) or supernatant (extracellular) in cells deprived of various indicated SG proteins. (H) depletion suppressed HCV core expression. Scale bars, 20 µm. (I) sirna reduces HCV subgenomic replicon activity. Error bars represent ± s.d. of triplicate experiments. * and **, P < 5 and 1, respectively. 10

11 A IKKα sipkr si si sipkr B C si Figure S7. Knockdown of various SG-associated proteins exhibited different effects on SG formation and aggregation. Huh cells were transfected with indicated sirnas for 3 days, and then infected with JFH-1 HCVcc for 2 days., IKK 11

12 α, SG markers and and were detected by immunofluorescence and examined by confocal microscopy. Magnifications of selected areas are shown in the right panels. (A) granules in -, -, or PKR-silenced cells. (B) in PKR-silenced cells. (C) in -silenced cells. Colors in far right pictures were changed for better visualization: (green instead of magenta) and (red). Scale bars, 20 µm. 12

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