NS5A basic cluster mutant has a defect in core—HCV RNA association.
- Publication date
- Publisher
Abstract
<p>(A) No impact of a Flag-tag inserted into core protein of a HCV reporter genome (JcR2a) on viral RNA replication and production of infectious virus particles. A schematic representation of the HCV genome indicating the insertion site in the core coding region (codon 2) is given in the top. Huh7-Lunet cells were transfected with the parental renilla luciferase reporter genome (JcR2a) or the variant containing a Flag-tag in core (JcRa2-Flag-core). Left panel: cells were lysed at given time points after transfection and replication was determined by measuring renilla luciferase activity. Right panel: culture supernatants of transfected cells harvested at given time points were used to infect naïve Huh7.5 cells and renilla luciferase activity was measured 72 h later. (B) Core protein amounts contained in transfected cells or released into culture supernatants 72 h after transfection are shown in the left panel (intra- and extracellular, respectively). Amounts of infectious virus released into culture supernatants and specific infectivity of released virus particles are given in the right panel. Mean and SEM of two independent experiments is shown. (C, D) Effect of mutations in NS5A on association of core protein with HCV RNA. Huh7-Lunet cells were transfected with HCV genomes specified in the bottom, 72 h later cells were lysed and Flag-tagged core was enriched by immunoprecipitation (IP) using a Flag-specific monoclonal antibody covalently linked to magnetic beads. (C) Efficiency of IP was determined by core-specific CMIA and normalized to the input. The non-tagged HCV genome (WT) served as technical control for specificity of immunoprecipitation. (D) HCV RNA and GAPDH mRNA (specificity control) coprecipitated with core protein was quantified by RT-qPCR and HCV RNA copy numbers (Y-axis in the left) or relative GAPDH mRNA values (Y-axis in the right) co-precipitated per pg core protein were calculated. Mean and SEM of ten independent experiments is shown. *, <i>p≤0</i>.<i>05; ***</i>, <i>p≤0</i>.<i>001</i>.</p