Fig. 4.

Autophagy machinery regulates translation and/or delivery of incoming viral RNA to the translation apparatus. Huh-7 cells were transduced with lentivirus expressing shRNA against Beclin-1, Atg4B, and GFP. (A) Analysis of JFH1 and Con1 neo/SGR replication. Neomycin selected cells were fixed and stained with Crystal Violet. A replication defective GND mutant (GDD-to-GND mutation in the NS5B protein) neo/SGR was used as a positive control for the neomycin selection. Results are representative of two independent experiments. (B) Analysis of Rluc/SGR translation/replication by monitoring Rluc activity at different times posttransfection. To assess the specificity of Rluc activity, control cells were treated with 5 μM of 2′-C-methyladenosine, a HCV polymerase inhibitor (denoted inh). For each independent experiment, Rluc activity was normalized to cell density and expressed as a percentage of that determined in control cells at 72 h posttransfection (mean ± SD; n = 4). (C) Translation of replication-deficient Rluc/SGR RNAs. Intracellular RNA levels and Rluc activity of replication deficient Rluc/SGR were determined at 6 h posttransfection. Rluc activities are statistically reduced in autophagy protein deficient cells compared to shRNA GFP expressing cells (P values <0.05 from paired Student's t test). In parallel transfections, Rluc activity expressed from pRL-TK plasmid (irrelevant-Rluc activity) was determined. For each independent experiment, Rluc activity was normalized to cell density and expressed as a percentage of that determined in control cells (mean ± SD; n = 3).