Gene Expression Control Using Hepatitis C NS3/4A Cis-Protease in Eukaryotic Systems

The precise control and engineering of dynamic gene expression programs in living
cells has long been a focus of synthetic biology. Here, we describe the development of
novel drug-inducible systems for regulating eukaryotic gene expression using anti-viral
protease inhibitors. First, we describe an approach in which the NS3/4A cis-protease
domain from Hepatitis C virus is used to control the subcellular localization (and thereby
function) of engineered transcription factors (TFs) over space and time. Using
constructs in which the TF is linked to a plasma membrane-tethering element via the
NS3/4A protease domain, we demonstrate that the timing and extent to which these
engineered TFs are able to gain access to the nuclear compartment can be finely tuned
via NS3/4A inhibition. Through fluorescence immunolocalization and quantitative/real-
time transcriptional activation assays, we demonstrate that these systems precisely
control the spatial distribution of the TFs with the use of FDA-approved NS3/4A
protease inhibitors, confining them to the cytoplasm or titrating their presence in the
nucleus.