The radical SAM enzyme, viperin, exerts a wide range of antiviral effects through both the synthesis of the antiviral nucleotide 3'-deoxy-3',4'-didehydro-CTP (ddhCTP) and through its interactions with various cellular and viral proteins. Here we investigate the interaction of viperin with hepatitis C virus nonstructural protein 5A (NS5A) and the host sterol regulatory protein, vesicle-associated membrane protein A (VAP-33). NS5A and VAP-33 form part of the viral replication complex that is essential for replicating the RNA genome of the hepatitis C virus. Using transfected enzymes in HEK293T cells, we show that viperin binds independently to both NS5A and the C-terminal domain of VAP-33 (VAP-33C) and that this interaction is dependent on the proteins being colocalized to the ER membrane. Coexpression of VAP-33C and NS5A resulted in changes to the catalytic activity of viperin that depended upon viperin being colocalized to the ER membrane. The viperin-NS5A-VAP-33C complex exhibited the lowest specific activity, indicating that NS5A may inhibit viperin's ability to synthesize ddhCTP. Coexpression of viperin with NS5A was also found to significantly reduce cellular NS5A levels, most likely by increasing the rate of proteasomal degradation. An inactive mutant of viperin, unable to bind the iron-sulfur cluster, was similarly effective at reducing cellular NS5A levels.