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, Silencer select siRNAs targeting MARCH8, RNA interference Custom Cherry-Pick ON-TARGETplus SMART pools siRNA library against E3 ligase genes and a non-targeting control (D-001810-10-05) were purchased from Dharmacon (Table S5)

;. Co-immunoprecipitations-co and . Neveu, All steps were done at 4 C. Membrane pellets were resuspended in 100 ml HME buffer. TDB buffer (2.5% Triton X-100, 25 mM triethanolamine-Cl (pH 8.6), 20 mM NaCl, 5 mM EDTA, 0.2% NaN3) was added to a final volume of 1 ml. Samples were incubated with anti-NS2 antibody for $2 hours and then overnight with protein A/G magnetic beads (Dynabeads, Life Technologies). Following PBS washes, bound proteins were eluted in SDS sample buffer, PBS and incubated with 1mM dithiobis-succinimidyl-propionate (DSP) crosslinker (Pierce) solution for 2 hours on ice (to allow covalent binding of the already bound interacting proteins). Tris (pH 7.5) was added at 20 mM for 15 minutes to quench unreacted DSP. Cells were washed once with PBS, resuspended in HME buffer (20 mM HEPES, 2012.

. Lindenbach, Luciferase activity in 20 ml of cell lysates was quantified as described above. Results represent log10 RLU values per 10 cm tissue culture dish. Intracellular infectivity assays 72 hr postelectroporation with HCV or DENV RNA, cells were trypsinized, collected by centrifugation, resuspended in 500 ml medium, lysed by freeze-thaw cycles, and pelleted twice at 3,650 3 g. Clarified supernatants diluted in complete medium were used to inoculate naive cells in triplicates, followed by lysis and luciferase assays at 72 hr. Results represent log10 RLU values per 10 cm tissue culture dish. Virus titration Extracellular and Intracellular titers were determined by limiting dilution assays based on immunohistochemical staining for core. 50% tissue culture infectious dose (TCID 50 ) was calculated, as described Lindenbach et al., 2005. Results are expressed as TCID 50 /ml. Minimal titers measured with the DE1-E2 mutant were used for background subtraction. RNA extraction and qRT-PCR Total RNA was isolated from cells, cell culture supernatants or gradient fractions using TRIzol (Invitrogen) or NucleoSpin RNA virus kit (Macherey-Nagel). qRT-PCRs mixtures were assembled in triplicates using High-Capacity RNA-to-cDNA and Power SYBR Green RNA-to-CT 1-Step Kit (Cat No:4389986: Applied Biosystems). The primers are listed in Table S6. Amplification and analysis were performed using StepOnePlus Real-Time-PCR system (Applied Biosystems). GAPDH was used as a control. Generation of MARCH8 knockout cell lines CRISPR guide RNA (gRNA) sequences were, RNA was synthesized from XbaI linearized J6/JFH (p7-Rluc2A) template using the T7 MEGAscript kit according to the manufacturer's protocol (Ambion), 2006.

. Ran, Viability assays Cells were incubated for 2 hours at 37 C with 10% alamarBlue reagent (TREK Diagnostic Systems). Fluorescence was detected by Tecan luminometer (Tecan) according to the manufacturers' protocols. NS2 protein expression and purification rNS2 (92-216 aa)-GST fusion was, Single clonal knockout of HEK293T and Huh7.5.1 cells were obtained using the PX458 vector that expresses Cas9 and sgRNA against MARCH8. Green fluorescent protein (GFP) positive single cells were sorted at 24 hours post-transfection using a BD InFlux Cell Sorter into 96-well plates and screened for knockout via Sanger sequencing and western blot, as described, 2013.

. Barouch-bentov, After 2-6 hour incubation at 37 C, the reaction was stopped by addition of SDS sample buffer. Detection of ubiquitination by IP Cells co-transfected with GLuc-NS2 and/or GLuc-MARCH8 or HCV RNA and control cells were treated for 2 hours with 10 mM MG-132 and lysed in a buffer containing 100 mM Tris-HCl, vitro ubiquitination assay 5 mg rNS2 was incubated with recombinant MARCH8, Huh7.5.1 cell extract, 2 mM ubiquitin-aldehyde, 0.5 mg/ml ubiquitin, 10 mM MG-132 and components of the ubiquitination kit (ENZO), including: E1 and E2 (UBE2H) enzymes and Mg-ATP in a final, vol.20, 2016.

, Dynabeads and 16 hour incubation at 4 C, wash with Catch and release IP wash buffer (EMD Millipore) supplemented with 2M Urea, and elution in X5 SDS sample buffer. Detection of ubiquitination using anti-K63 TUBE technology Cells co-expressing GLuc-NS2 and GLuc-MARCH8 were treated with 10 mM MG-132 for 2 hours, washed with PBS, and lysed in the lysis buffer described above plus 500 nM FLAGâ Anti-K63 TUBE reagent (LifeSensors), Anti-NS2 or IgG antibodies were then added to the clarified supernatants for $2 hours, vol.100

, NS2 stability assays 72 hours post-transfection of MARCH8 KO and WT Huh7.5.1 cells with HCV RNA J6/JFH, cells were treated with cycloheximide (100 mg/ml) for 8 hours and samples were collected every 2 hours. NS2 expression was analyzed in cell lysates via western blots and quantified by imageJ (NIH). The half-life of, Triton X-100, and DUB inhibitors, and incubated for 1 hour on ice. IP with anti-NS2 antibody was conducted as described above