NS5A is a large hydrophilic phosphoprotein that is essential for the HCV life cycle and is found in association with virus-induced membrane vesicles, termed the membranous web. NS5A is a proline-rich protein composed of approximately 447 amino acids, which is divided into three domains. These domains are linked by two low-complexity sequences that are either serine- or proline-rich. Domain I is a zinc binding domain and X-ray crystallography studies indicated alternative dimer conformations of domain I of NS5A. Domain II and III are unstructured, shown by NMR studies. Domain I is preceded by an N-terminal amphipathic helix which allows the protein to associate with endoplasmic reticulum-derived membranes. Although X-ray crystallographic studies revealed dimer conformations of NS5A domain1, recent in solution structural characterization studies showed that NS5A proteins form higher-order structures by dimeric subunits of NS5A domain 1. Moreover, the overall structural model of NS5A highlights the variability of intrinsic conformations of the D2 and D3 domains between HCV genotypes. Therefore, it is still under debate which conformation\s of NS5A is functional and also targeted by NS5A inhibitors.
NS5A mainly exists in two distinct phosphorylated forms, a hypophosphorylated and a hyperphosphorylated form, but the exact function of the phosphorylation has not been determined.