The NS4A Y45F mutation results in production of partially formed virions.

Supernatants harvested from Huh7.5 cells 48 hours post-electroporation of NS4A WT, NS4A Y45F, or ΔE1/E2 in vitro transcribed HCV RNA (JFH1) were analyzed for HCV RNA by RT-qPCR (A) or Core protein by ELISA (B). Data in A are presented as mean ± SEM (n = 3) and analyzed by unpaired t-test. ****Pin vitro transcribed HCV WT or NS4A Y45F RNA (JFH1) were concentrated, fractionated over a 10–50% iodixanol gradient, and collected into 10 equal fractions. Fractions were analyzed by focus-forming assay for infectivity, represented as FFU/mL, and RT-qPCR for HCV RNA, shown as a measure of copy number (C) and by immunoblot for Core (D). In the bottom panels of the immunoblots, fractions were treated with 2-mercaptoethanol and DTT and then boiled before immunoblotting for anti-Core. Fractions 1–10 correspond with fractions running from top to bottom of the gradient, with the densities listed at the bottom. Data in C is presented as mean ± SD (n = 3); C-D are representative of 2 independent experiments.