Probing the relationship between RNA-stimulated ATPase and helicase activities of HCVNS3 using 2 '-O-methyl RNA substrates

The hepatitis C virus (HCV) NS3 protein contains an amino terminal protease (NS3 aa. 1-180) and a carboxyl terminal RNA helicase (NS3 aa. 181-631). NS 3 functions as a heterodimer of NS3 and NS4A (NS3/4A). NS3 helicase, a nucl eic acid stimulated ATPase, can unwind RNA, DNA, and RNA:DNA duplexes, prov ided that at least one strand of the duplex contains a single-stranded 3' o verhang (this strand of the duplex is referred to as the 3' strand). We hav e used 2'-O-methyl RNA (MeRNA) substrates to study the mechanism of NS3 hel icase activity and to probe the relationship between its helicase and RNA-s timulated ATPase activities. NS3/4A did not unwind double-stranded (ds) MeR NA. NS3/4A unwinds hybrid RNA:MeRNA duplex containing MeRNA as the 5' stran d but not hybrid duplex containing MeRNA as the 3' strand. The helicase act ivity of NS3/4A was 50% inhibited by 40 nM single-stranded (ss) RNA but onl y 35% inhibited by 320 nM ss MeRNA. Double-stranded RNA was 17 times as eff ective as double-stranded MeRNA in inhibiting NS3/4A helicase activity, whi le the apparent affinity of NS3/4A for ds MeRNA differed from ds RNA by onl y 2.4-fold. However ss MeRNA stimulated NS3/4A ATPase activity similar to s s RNA. These results indicate that the helicase mechanism involves 3' to 5' procession of the NS3 helicase along the 3' strand and only weak associati on of the enzyme with the displaced 5' strand. Further, our findings show t hat maximum stimulation of NS3 ATPase activity by ss nucleic acid is not di rectly related to procession of the helicase along the 3' strand.