1. M 30’ 60’ - Proteinase K+ Proteinase K core actin B. b a S1 10 0 1 2 3 4 FL1-H HCVne: 17 - 21% A. a 10 0 1 2 3 4 FL1-H HCVne den: 0.9 - 2% b FIGURE S1: HCVne particles enter T lymphocytes. A) FACS analysis of intracellularly core- immumolabeled Jurkat cells maintained in serum-free conditions that were incubated with (a) HCVne (40ng of core protein, represented with dotted line) or treated with (b) the same concentrations of the HCVne fraction heat denatured (40min at 95 o C), for 40 min at 4 o C and 60min at 37 o C. Before immunolabeling, all samples were treated with 50μgr/ml of proteinase K for 20 min at 4 o C for the digestion of bound particles. In both histogram plots, the grey-shaded curve represents mock treated cells. B) Lysates from Jurkat cells incubated with HCVne (40ng of core protein) for 30 min or 60min at 37 o C and either further treated with proteinase K (+) or not (-). Western blot with polyclonal anti- core (a) and monoclonal anti-actin (b) are presented.

2. c p38 0 1 2 3 4 5 6 7 8 9 10 mock+30'1h2h6h12h t.p.inc fold induction HCVne 1746 S2 (+) mock 30’ 1h 2h 6h 12h 1h 6h 12h HCVne 1746 pp38 p38 b a FIGURE S2: HCVne particles mediate activation of MAPK-p38 in Jurkat cells. Time course of p38 phosphorylation from serum starved Jurkat cell lysates incubated with HCVne particles (15ng of core protein) for 30min, 1, 2, 6 and 12 hours at 37 o C. Anisomycin treated cells (10μM) were used as a positive (+) control. Cells treated with a fraction of equivalent sucrose density from Sf9 cell lysates infected with Bac1746 were used as negative controls. Western blot (representative experiment of triplicates) with phospho-p38 (a), p38 (b), and quantification of the optical densities of phospho-p38 immunoreactive bands normalized to the optical densities of total p38 in the same samples (c) are presented.

3. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 mock+30'1h2h 6h12h24h2h12h 24h t.p.inc. fold activation - SB203580 + SB203580 HCVne den HCVne IL-2 S3 A. B. IL-2 28S (+) 6h 12h 24h 48h 72h 6h 12h 24h HCVne den HCVne a IL-2 ELISA 0 50 100 150 200 250 mock+2h6h8h12h18h24h t.p.inc. IL-2 conc (pg/ml) HCVne 1746 b C. IL-2 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 + 6h12h 24h 48h 72h t.p.inc. fold activation HCVne den HCVne

4. FIGURE S3: Uptake of HCVne particles leads to IL-2 transcriptional activation and secretion. A) Total mRNAs were isolated from serum starved Jurkat cells that were stimulated with PMA/ ionomycin for 4 hours, subsequently incubated with HCVne particles for 6, 12, 24, 48 and 72 hours at 37 o C and were subjected to RT-PCR. As negative controls, heat denatured HCVne (95 o C for 40 min) were used. PCR was performed with specific primers for IL-2 (a) and 28S RNA (b). All samples were compared with the stimulated positive sample (+), where no HCVne were subsequently added. The same experiment was repeated 3 times and a representative image is presented. Densitometric analysis after normalisation is presented in arbitrary units (b). Samples treated with HCVne, as well as the positive sample, are represented in dark grey bars and negative controls are represented in light grey bars. B) Comparison of IL-2 transcriptional activation after HCVne incubation in serum starved Hut-78 cells that were either pretreated or not pretreated with SB203580. Samples that were not pretreated with the p38 inhibitor are represented in dark grey bars while the pretreated ones are represented in light grey bars. Positive (PMA/ ionomycin) and negative controls (den HCVne), are also presented. C) Serum-starved Jurkat cells that were stimulated with PMA/ ionomycin for 3 hours, subsequently challenged with HCVne for 2, 6, 12, 18, 24 hours at 37 o C and IL-2 ELISA was performed in their supernatants. The levels of secreted IL-2 are measured in pgr/ml. Supernatants from cells incubated with a corresponding fraction from Bac 1746 were used as negative controls. Samples treated with HCVne, as well as the stimulated positive sample (+), are represented in dark grey bars and negative controls are represented in light grey bars. All ELISA test were performed in duplicates and repeated twice.

5. FIGURE S4: FACS analysis of human liver-infiltrated T cells maintained in 1% FCS incubated with (a) HCVne (40ng of core protein represented with continuous line) or (b) heat denatured (40min at 95 o C) HCVne for 40min at 4 o C and 1 hour at 37 o C and intracellularly immunolabeled with core antibody. In both histogram plots, the grey-shaded curve represents mock treated cells immunolabeled with core antibody.