Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein

During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.