Control of innate immune signaling and membrane targeting by the hepatitis C virus NS3/4A protease are governed by the NS3 Helix α ₀

Hepatitis C virus (HCV) infection is sensed in the host cell by the cytosolic pathogen recognition receptor RIG-I. RIG-I signaling is propagated through its signaling adaptor protein MAVS to drive activation of innate immunity. However, HCV blocks RIG-I signaling through viral NS3/4A protease cleavage of MAVS on the mitochondrion-associated endoplasmic reticulum (ER) membrane (MAM). The multifunctional HCV NS3/4A serine protease is associated with intracellular membranes, including the MAM, through membrane-targeting domains within NS4A and also at the amphipathic helixα ₀ of NS3. The serine protease domain of NS3 is required for both cleavage of MAVS, a tail-anchored membrane protein, and processing the HCV polyprotein. Here, we show that hydrophobic amino acids in the NS3 helixα ₀ are required for selective cleavage of membrane-anchored portions of the HCV polyprotein and for cleavage of MAVS for control of RIG-I pathway signaling of innate immunity. Further, we found that the hydrophobic composition of NS3 helixα ₀ is essential to establish HCV replication and infection. Alanine substitution of individual hydrophobic amino acids in the NS3 helixα ₀ impaired HCV RNA replication in cells with a functional RIG-I pathway, but viral RNA replication was rescued in cells lacking RIG-I signaling. Therefore, the hydrophobic amphipathic helix α ₀ of NS3 is required for NS3/4A control of RIG-I signaling and HCV replication by directing the membrane targeting of both viral and cellular substrates.