ns3 from hepatitis c virus strain jfh1 is an unusually robust helicase that is primed to bind and unwind viral rna hepatitis c viruses hcv encode a helicase enzyme that is essential for viral replication and assembly nonstructural protein 3 ns3 this helicase has become the focus of extensive basic research on the general helicase mechanism and it is also of interest as a novel drug target despite the importance of this protein mechanistic work on ns3 has been conducted almost exclusively on variants from hcv genotype 1 our understanding of ns3 from the highly active hcv strains that are used to study hcv genetics and mechanism in cell culture such as jfh1 is lacking we therefore set out to determine whether ns3 from the replicatively efficient genotype 2a strain jfh1 displays novel functional or structural properties using biochemical assays for rna binding and duplex unwinding we show that jfh1 ns3 binds rna much more rapidly than the previously studied ns3 variants from genotype 1b unlike ns3 variants from other genotypes jfh1 ns3 binds rna with high affinity in a functionally active form that is capable of immediately unwinding rna duplexes without undergoing ratelimiting conformational changes that precede activation unlike other superfamily 2 sf2 helicases jfh1 ns3 does not require long 3 overhangs and it unwinds duplexes that are flanked by only a few nucleotides as in the folded hcv genome to understand the physical basis for this we solved the crystal structure of jfh1 ns3 revealing a novel conformation that contains an open positively charged rna binding cleft that is primed for productive interaction with rna targets potentially explaining robust replication by hcv jfh1importance genotypes of hcv are as divergent as different types of flavivirus and yet mechanistic features of hcv variants are presumed to be held in common one of the most wellstudied components of the hcv replication complex is a helicase known as nonstructural protein 3 ns3 we set out to determine whether this important mechanical component possesses biochemical and structural properties that differ between common strains such as those of genotype 1b and a strain of hcv that replicates with exceptional efficiency jfh1 classified as genotype 2a indeed unlike the inefficient genotype 1b ns3 which has been well studied jfh1 ns3 is a superhelicase with strong rna affinity and high unwinding efficiency on a broad range of targets crystallographic analysis reveals architectural features that promote enhanced biochemical activity of jfh1 ns3 these findings show that even within a single family of viruses drift in sequence can result in the acquisition of radically new functional properties that enhance viral fitness