Interactions of the Disordered Domain II of Hepatitis C Virus NS5A with Cyclophilin A, NS5B, and Viral RNA Show Extensive Overlap

Abstract

Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5A^DII) is largely disordered, which makes it difficult to characterize the protein-protein or protein-nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5A^DII and its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5A^DII. These experiments have led to the identification of an RNA binding motif (³⁰⁵RSRKFPR³¹¹) in an arginine-rich region of NS5A^DII. Furthermore, we show that K308 is indispensable for both RNA and NS5B binding, whereas W316, further downstream, is essential for protein-protein interactions with CypA and NS5B. Most importantly, NS5A^DII binding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5A^DII modulates the activity of NS5B and potentially regulates HCV replication.