The interaction between the hepatitis C proteins NS4B and NS5A is involved in viral replication.

Hepatitis C virus (HCV) replicates in membrane associated, highly ordered replication complexes (RCs). These complexes include viral and host proteins necessary for viral RNA genome replication. The interaction network among viral and host proteins underlying the formation of these RCs is yet to be thoroughly characterized. Here, we investigated the association between NS4B and NS5A, two critical RC components. We characterized the interaction between these proteins using fluorescence resonance energy transfer and a mammalian two-hybrid system. Specific tryptophan residues within the C-terminal domain (CTD) of NS4B were shown to mediate this interaction. Domain I of NS5A, was sufficient to mediate its interaction with NS4B. Mutations in the NS4B CTD tryptophan residues abolished viral replication. Moreover, one of these mutations also affected NS5A hyperphosphorylation. These findings provide new insights into the importance of the NS4B-NS5A interaction and serve as a starting point for studying the complex interactions between the replicase subunits.