Proper insertion of V5-B2 was verified through orientation PCR and sequencing. Infectious virus was produced by electroporation of linearized plasmid as described previously [46, 47]. Electroporations were performed in BHK-21 cells and each virus was passaged once in Vero cells. All viruses were Autophagy signaling inhibitor aliquoted, titrated using standard assays, and maintained at -80°C until use. Immunoblot analysis For immunoblot analysis, cell monolayers were infected with TE/3’2J, selleck screening library TE/3’2J/GFP, and TE/3’2J/B2 virus at a MOI~0.01, or mock-infected with medium. Forty-eight hours post-infection, medium was removed and cells were scraped

into PBS containing protease inhibitors (Roche Applied Science, Indianapolis, IN). Cell suspensions were sonicated and stored at -20°C. Ten micrograms of total protein were separated by SDS-polyacrylamide gel electrophoresis in a 10% gel and transferred to a nitrocellulose membrane at 30 volts. Membranes were blocked for 1 hour at room temperature in PBS plus 0.05% Tween-20 (PBS-T) and 5% lowfat dry milk (blocking buffer). V5-B2 protein was detected by incubating membranes at 4°C overnight with a mouse anti-V5 IgG antibody (Invitrogen Corporation, Carlsbad, CA) diluted 1:5,000 in blocking buffer followed by a room temperature incubation with a horseradish peroxidase-conjugated Temsirolimus goat anti-mouse IgG secondary antibody (KPL, Inc.,

Gaithersburg, MD) diluted 1:1,000 in blocking buffer for 30 minutes. The Pierce ECL western detection kit (Thermo Fisher Scientific, Inc., Rockford, IL) was used to develop the membranes according to manufacturer’s protocols. Chemiluminescence was detected using the Storm 860 phosphoimager

(Molecular Dynamics, Inc., Sunnyvale, CA). In vitro dicing assay Cell-free lysates were generated from Aag2 cells that Cytidine deaminase were mock-infected or infected with TE/3’2J, TE/3’2J/GFP, or TE/3’2J/B2 virus (MOI: 0.01). Lysates were formed 36 hours post-infection using a protocol modified from Haley et al [49]. Briefly, cells were washed three times in PBS and resuspended in 1× lysis buffer (100 mM potassium acetate; 30 mM Hepes-KOH, pH 7.4; 2 mM magnesium acetate) with protease inhibitors and 5 mM DTT. The cells were disrupted in a Dounce homogenizer and centrifuged at 14,000 × g for 25 minutes at 4°C. The supernatant was flash frozen in a dry ice/ethanol bath and stored at -80°C. Dicing activity reactions were constituted as described previously [49] and incubated at 28°C. Each reaction contained 1/2 volume of cell lysate (normalized for protein concentration), 1/3 volume of 40× reaction mix (50 μl water; 20 μl 500 mM creatine monophosphate; 20 μl amino acid stock at 1 mM each, 2 μl 1 M DTT, 2 μl 20 U/μl RNasin, 4 μl 100 mM ATP, 1 μl 100 mM GTP, 6 μl 2 U/μl creatine phosphokinase, 16 μl 1 M potassium acetate) and 450 ng of 500 bp biotinylated β-gal dsRNA [49].