The level of expression of Spn4A variants was first optimized by infecting human hepatoma Huh-7.5.1 cells (highly permissive for HCV JFH-1 infection) with different multiplicity of infection (moi) of recombinant adenovirus expressing intracellularly retained Spn4A.RRLL(r). Cellomics HCS analysis revealed that over 90% of these Huh-7.5.1 cells expressed Spn4A.RRLL(r) at a moi of 50 ( Figure S1 ). Cellomics HCS was also used to determine if cell death occurs following 2 days of pre-treatment with Spn4A.RRLL(r) or Spn4A.RRLL(s) compared to the control (Ad-Empty) followed by 72 hours of HCV infection as employed in the experiments below ( Figure S2 ). We observed no significant reductions in total cell numbers under these experimental conditions. The high-content analysis of cell death was confirmed using an MTS-based cell viability assay (data not shown). The lack of detectable toxicity induced by Ad-Spn4A.RRLL(r) and Ad-Spn4A.RRLL(s) up to a moi of 50 showed that these variants can be tested over a very wide dynamic range in hepatoma cells.