Mutations affecting the NS5A basic cluster reduce interaction with HCV RNA.

Abstract

<p>(A) Schematic representation of the Jc1 genome containing a Flag-tag in NS5A DII. Huh7-Lunet cells were transfected with HCV genomes specified in the bottom and 72 h later cells were lysed and NS5A was enriched by immunoprecipitation (IP) using a Flag-specific monoclonal antibody covalently linked to magnetic beads. Captured NS5A proteins were separated by electrophoresis into an 8% acrylamide gel and analyzed by Western blot using a mono-specific NS5A antibody. The same amounts of input proteins were loaded onto the gel in parallel. The efficiency of the Flag-NS5A IP was determined by quantifying the bands and normalizing to the input signals. The non-tagged HCV genome (WT) served as technical control for specificity of immunoprecipitation. (B) Quantification of HCV RNA co-precipitated with Flag-NS5A. HCV RNA and GAPDH mRNA (specificity control) was determined by RT-qPCR. The percentage of viral RNA copies co-precipitated (co-IP) with NS5A was determined and used to calculate the enrichment of HCV RNA co-precipitated with NS5A. Note that GAPDH mRNA was below the detection limit in the co-precipitated samples and therefore is not displayed. Mean and SEM of six independent experiments is shown. *, <i>p ≤0</i>.<i>05; **</i>, <i>p ≤0</i>.<i>01; not significant (NS)</i>, <i>p ≥0</i>.<i>05</i>.</p