NS5A basic cluster mutant is impaired in core envelopment.

<p>(A) Huh7-Lunet cells were transfected with RNA genomes specified in the top: Jc1 wildtype (WT), NS5A R352-355E basic cluster mutant (BCM) and NS5A serine cluster mutant S452/454/457A (SCM). Twelve, 24 and 48 h post transfection cell lysates were prepared and separated by using a 0–30% linear sucrose gradient (left, middle and right panel, respectively). Core protein amounts contained in each fraction were quantified and normalized to total core contained in all fractions. (B) The amounts of E2 (left), NS5A (middle) and ADRP (right) contained in each fraction of cell lysates prepared 48 h post transfection were determined by quantitative Western blot. For each fraction, relative protein amounts are displayed (lower panels). Mean and SEM of two independent experiments are shown. (C) Huh7-Lunet cells were transfected with given HCV genomes and 48 h later cell lysates were prepared and either mock treated or incubated with 15 μg/ml proteinase K (PK) for 40 min on ice. As positive control, samples were treated with 1% Triton X-100 (TX-100) prior to PK digestion. The amount of core protein resistant to PK treatment was determined by Western blot and CMIA (left and right panel, respectively). The graph shows the percentage of PK-resistant core protein. Mean and SEM of three independent experiments is shown. **, <i>p ≤0</i>.<i>01; ***</i>, <i>p ≤0</i>.<i>001</i>.