FIG 2 .

NS2 harbors two dileucine motifs that mediate AP binding and HCV release. (A) Dileucine motifs in the C terminus of NS2 and the cloned mutations. The schematic shown was based on data from reference 46. (B) Levels of NS2 in lysates of cells transfected with the plasmids indicated and blotted with anti-GLuc and anti-actin antibodies. (C) Interactions of WT or mutant NS2 with AP-1A, AP-1B, and AP-4 by PCAs. Plotted are NLRs relative to WT NS2-AP binding. (D) Cells were electroporated with WT or mutated NS2 bicistronic J6/H77NS2/JFH HCV RNA. HCV RNA replication measured via luciferase assays 8 and 72 h after HCV RNA electroporation. RLU, relative light units. (E) HCV infectivity measured via luciferase assays by inoculating naive cells with lysates (intracellular) and supernatants (extracellular) from electroporated cells. (F) Intra- and extracellular infectivity titers measured by limiting-dilution assays (top) and percentages of intracellular infectivity per total (intra- and extracellular) infectivity for the dish (bottom). Viral RNA (G) and HCV core protein (H) release into culture supernatant at 72 h postelectroporation measured by qRT-PCR and ELISA, respectively. GNN is a replication-incompetent HCV strain. ΔE1-E2 is an assembly-defective mutant. Results in panels C to H represent data pooled from at least two independent experiments each with 3 to 10 biological replicates. Shown are the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (relative to the corresponding WT; one-way [C and E to H] or two-way [D] ANOVA with Dunnett’s post hoc test).