Vivaspin

The urine samples of patients and controls were concentrated 60 times using Vivaspin (Millipore Corporation, Massachusetts, USA). Urinary levels of HMGB1 were tested using commercially available ELISA kits (Shino-TEST). The assay was conducted according to the manufacturer’s instructions. Urinary HMGB1 was expressed as HMGB1/Cr ratio (mg/μmolCr) to correct for differences in dilution.

The lectin CCL2 was expressed using a pET22b vector as described earlier (Schubert et al. 2012 (link)). Either Luria broth (Thermo Fisher Scientific) or M9 minimal medium (Sambrook 2001 ) with or without ¹³C and ¹⁵N isotope-labeling was used as culture medium. After affinity chromatography purification the buffer was exchanged to 50 mM KH₂PO₄/K₂HPO₄ pH 5.8, 150 mM NaCl by dialysis (3.5 kDa cutoff, Spectra/Por) and the proteins were concentrated with ultrafiltration devices (3 kDa cutoff, Amicon/Millipore or Vivaspin/Satorius). Most CCL2 spectra were recorded without ligand, but few were in complex with the trisaccharide GlcNAcβ1,4[Fucα1,3]GlcNAcβO(CH₂)₅COONa at pH 4.7. The individual domains of the RNA-binding protein hnRNP A1 were expressed and purified as described previously (Barraud and Allain 2013 (link)). Both domains were individually studied in complex with RNA, the RNA-recognition motif 1 (RRM1) in complex with the RNA UUAGGUC and RRM2 with the RNA UCAGUU in 10 mM NaH₂PO₄/Na₂HPO₄ pH 6.5 as described earlier (Beusch et al. 2017 (link)). The tandem zinc-knuckles of Lin28 (amino acids 124–186) were expressed, purified and complexed with AGGAGAU RNA from pre-miRNA let-7 as described (Loughlin et al. 2012 (link)). Spectra of the Lin28-RNA complex were measured in 10 mM sodium acetate pH 5.6, 1.5 mM β-mercaptoethanol and 0.15 mM ZnCl₂ at 303 K.

10 nm amino-PEG functionalized GNPs were synthesized according to reference [24 (link)]. Briefly, HAuCl₄ (Sigma Aldrich, Overijse, Belgium) and TA-PEG₅₅₀-OCH₃ (Biochempeg Scientific Inc., Watertown, MA, USA) were mixed at a 2000:1 Au: PEG molar ratio in deionized water and stirred at room temperature for 1 h. NaBH₄ (Sigma Aldrich) was then added to the mixture under vigorous stirring and the solution was left stirring for 3 h. Then, TA-PEG₄₀₀-NH₂ (Biochempeg Scientific Inc., Watertown, MA, USA) was added to the solution for extra passivation. After 3 h of stirring, the colloidal suspension was purified using a membrane filtration device (Vivaspin, Millipore, Darmstadt, Germany).
GNPs were lyophilized with a freeze-drying system (Alpha 2-4 LD Plus; Analis, Rhisnes, Belgium) and stored at 4 °C for further use. In all experiments, cells were incubated with 50 µg of gold per mL of medium, which corresponds to 8.22 nM of GNPs.

In vitro transcription and RNA electroporation were performed as previously described [48 (link)]. Approximately 3 × 10⁶ Huh7.5.1 cells were electroporated with in vitro-transcribed RNAs derived from JC1 or JFH1-ad34-5A-Rluc containing the cell culture adapted mutations in the E2 and p7 proteins [45 (link)]. The culture supernatants were collected at 3–5 days after electroporation, and naïve Huh7.5.1 cells were infected with the generated viruses. The cell culture media of infected cells were collected at 3–5 days after infection. The HCV-containing supernatants were then filtered through with a 0.45 μm filter, and the filtrate was concentrated with a Vivaspin (100-kD cut-off; Millipore). The concentrated HCV culture medium was loaded onto a 20% sucrose cushion, and HCV particles were collected by ultracentrifugation at 4°C for 4 h at 36,000 rpm (SW41 rotor; Beckman). The viral pellets were resuspended with complete medium. Regarding the transient replication assay, in vitro transcripts of HCV subgenomic replicons were generated, purified, and electroporated into Huh7-Lunet/T7 cells. Firefly luciferase activities in cell lysates were measured as previously described [49 (link)].

Liposomes were prepared using the sonication method, as previously reported24 (link). Briefly, a lipid film was prepared by evaporating a chloroform solution containing the corresponding lipids in the desired proportion (Supplementary Table 1). The resulting film was hydrated with PBS (typically 10 ml) and sonicated for 25 min using a 150 V/T Ultrasonic Homogenizer (Biologics, Inc., Ramsey, NJ) working at 30% power output. After quick centrifugation, size and Z-potential measurements were performed on a NanoSeries Z-Sizer (Malvern Instruments, Malvern, UK) and a Zeta PALS analyser (Brookhaven Instruments Corporation, Holtsville, NY), respectively. Liposomes containing DSPE-DFO were concentrated using a 100-kDa VivaSpin (Millipore, Billerica, MA) tube and washed twice with PBS.