Recent Progress in Structures and Functions of Hepatitis C Virus NS3/4A Proteins

Hepatitis C virus (HCV) chronically infects over 50 million people worldwide and poses a significant risk to global health. The HCV NS3/4A complex, a bifunctional enzyme comprising a protease and a helicase domain, is indispensable for viral replication and immune evasion, making it a pivotal target for direct-acting antiviral agents (DAAs). Here, we summarize its structural features, functional mechanisms, and implications in drug design and protein engineering (e.g., nanopore sequencing applications). The NS3 protease domain is activated by the NS4A cofactor, which mediates viral polyprotein processing and relies on a zinc-binding site for structural stability. The C-terminal helicase domain catalyzes ATP-dependent 3'→5' unwinding, and allosteric crosstalk between the protease and helicase domains dynamically modulates the enzymatic activity, balancing unwinding velocity and processivity. Beyond supporting viral replication, NS3/4A cleaves MAVS to abolish RIG-I/MDA5 signaling but spares TRIF, leaving TLR3-mediated immunity intact; it also modulates host lipid and iron metabolism, contributing to HCV pathogenesis. Notably, structural and functional studies of NS3/4A lay a solid theoretical foundation for developing novel therapeutic strategies. Currently, DAAs targeting NS3/4A have achieved high sustained virologic response rates; however, resistance-associated substitutions remain a major clinical challenge, particularly in genotype 3 infections. Emerging therapeutic strategies targeting NS3/4A include allosteric inhibition and proteolysis-targeting chimeras (PROTACs)-mediated degradation.