Hepatitis C virus (HCV) is a hepatotropic pathogen present in approximately 2.8% of the population with 3-4 million new infections every year. Up to 85% of infections progress to a chronic disease associated with development of steatosis, cirrhosis and hepatocellular carcinoma. Viral protein, Nonstructural 5A (NS5A), has eluded a defined role in the HCV life cycle despite being essential to viral propagation and host cell modulation. NS5A is a phosphoprotein appearing as two molecular weight proteoforms by SDS-PAGE referred to as the basally phosphorylated and hyperphosphorylated forms. Different NS5A phospho-proteoforms may direct its function acting as a molecular switch between replication and assembly. Two aspects of NS5A biology were addressed: 1) NS5A host cell protein-protein interactions and 2) defining phosphorylation sites on NS5A. To identify NS5A-host protein-protein interactions, a novel tandem affinity purification (TAP) technique was implemented. A HBH (histidine-biotin-histidine) tag was affixed to NS5A-2a (JFH1) and cells were generated stably expressing this construct. Tagged proteins were purified using native-state (nTAP) and cross-linked, denaturing (xdTAP) using immobilized metal chelate and streptavidin resins. Purified samples were subjected to tandem-mass spectrometry and database searching to generate an NS5A interacting protein list. Co-immunoprecipitation and colocalization confirmed host cell Cell Cycle and Apoptosis Regulator protein 2 (CCAR2) as an NS5A interacting protein. Phosphorylation analysis used NS5A isolated as a tagged protein, part of a subgenomic (SG) replicon, or a tagged protein within a SG replicon. Subsequent tandem mass spectrometry allowed for identification of 28 phosphorylation sites, 20 of which were novel. Phosphoablatant and phosphomimetic mutation of the phosphoacceptor sites were used to evaluate the impact of phosphorylating these residues to the JFH1 virus. While majority of these mutations had no impact, T204D, T210A/D, S225D, S229A/D, S232A/D, S235A/D, S238A/D, T334A/D, and T363A/D mutants were reduced in their replicative capacity. NS5A phospho-proteoform ratios were evaluated with S151D, S225A and S232A mutants primarily basally phosphorylated while T213D, T210A/D were mainly hyperphosphorylated. Mutants T210D, S229A, S229D, and S235A failed to produce virus. A novel observation was made that NS5A is principally hyperphosphorylated at early time-points post RNA electroporation but past 72 hours the basally phosphorylated form predominates.