Treatment of hepatitis C patients with direct-acting antiviral drugs involves 
the combination of multiple small-molecule inhibitors of distinctive mechanisms 
of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific 
for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon 
cells and was active in genotype 1 HCV-infected patients in a proof-of-concept 
clinical trial (1). Here, we describe a potential mechanism of action (MoA) 
wherein ACH-806 alters viral replication complex (RC) composition and function. 
We found that ACH-806 did not affect HCV polyprotein translation and processing, 
the early events of the formation of HCV RC. Instead, ACH-806 triggered the 
formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in 
replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production 
was negatively regulated by NS3, and its appearance in turn was associated with 
reductions in NS3 and, especially, NS4A content in RCs due to their accelerated 
degradation. A previously described resistance substitution near the N terminus 
of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A 
conferred by ACH-806 treatment. Taken together, we show that the compositional 
changes in viral RCs are associated with the antiviral activity of ACH-806. 
Small molecules, including ACH-806, with this novel MoA hold promise for further 
development and provide unique tools for clarifying the functions of NS4A in HCV 
replication.