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Background:  We have set up a system to examine natural variants arising during acute human infection in order to address the hypothesis that hepatitis C virus (HCV) sequence variation is constrained by virologic fitness. HCV exists as an array of closely related but distinct viral sequence variants, or quasi-species. Previous studies have examined the effects of divergent sequences on HCV-specific immune response, but little is known about the effects of mutation on viral protein functions. We have studied the activity of the helicase, which is contained in the 3’ portion of the NS3 gene. The helicase activity unwinds double stranded and highly structured RNA (and DNA) and is essential for productive infection. NS3 helicase activity from multiple sources has not been examined.

Methods:  Serum was obtained from injection drug users during acute HCV infection. The NS3 helicase domain was cloned and expressed in an E. coli expression system. His-tagged helicase was purified by HPLC on a Ni²⁺ column. Helicase activity was measured as reduction in signal due to unwinding of labeled paired oligonucleotides, one of which is immobilized.

Results:  Purification of helicase resulted in a single band on Coomassie-stained SDS-PAGE. Helicase activity reached a maximum of 89% unwinding at 8 µg/mL helicase. This activity was present at 37° and 42°, but was reduced by pH ≥ 7.6 and high potassium concentration.

Conclusions:  The HCV NS3 helicase protein was successfully cloned from an acute subject, and its activity has been characterized. Its activity was consistent with prior reports from a single strain. The establishment of this system enables sequence-specific comparison of helicase activity from primary isolates. Studies of the effects of sequence variation on helicase activity will provide insight regarding the impact of immune escape on enzymatic function, as well as highlight specific domains that may be required for function that are as yet unknown. These studies may also become increasingly relevant as helicase inhibitors are currently being developed as potential antiviral agents for the treatment of HCV infection, and they will be expected to select for inhibitor-resistant sequences.

Keywords: HCV; NS3 Helicase; mutation