The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

Abstract

The nonstructural 3 (NS3) protein encoded by the hepatitis C virus possesses both an N-terminal serine protease activity and a C-terminal 3 -5 helicase activity. This study examines the effects of the protease on the helicase by comparing the enzymatic properties of the full-length NS3 protein with truncated versions in which the protease is either deleted or replaced by a polyhistidine (His tag) or a glutathione S-transferase fusion protein (GST tag). When the NS3 protein lacks the protease domain it unwinds RNA more slowly and does not unwind RNA in the presence of excess nucleic acid that acts as an enzyme trap. Some but not all of the RNA helicase activity can be restored by adding a His tag or GST tag to the N terminus of the truncated helicase, suggesting that the effects of the protease are both specific and nonspecific. Similar but smaller effects are also seen in DNA helicase and translocation assays. While translocating on RNA (or DNA) the full-length protein hydrolyzes ATP more slowly than the truncated protein, suggesting that the protease allows for more efficient ATP usage. Binding assays reveal that the full-length protein assembles on single-stranded DNA as a higher order oligomer than the truncated fragment, and the binding appears to be more cooperative. The data suggest that hepatitis C virus RNA helicase, and therefore viral replication, could be influenced by the rotations of the protease domain which likely occur during polyprotein processing.The epidemic caused by infection by the hepatitis C virus (HCV) 1 is still a global crisis despite recent therapeutic advancements (1). Because HCV cannot be conventionally cultivated in cell culture and the only other host is the chimpanzee, the enzymes encoded by HCV have been studied intensely as targets for rational drug design. One key viral enzyme is the multifunctional nonstructural protein 3 (NS3), which possesses a serine protease activity and an ATPase function that fuels the ability of the protein to unwind RNA and DNA duplexes. Although it is clear that both the protease and helicase functions are necessary for viral replication (2), it is not clear whether the two functions, which reside in independent protein domains, cooperate in any manner (3).To examine possible effects of the NS3 protease on its helicase function, the activities of the full-length NS3 protein were rigorously compared with the same protein lacking the protease and also with recombinant proteins in which the protease is replaced with other non-HCV peptides. The experiments were designed to uncover effects of the NS3 protease domain on its helicase function which are either specific or nonspecific. Nonspecific effects are defined as those that can be duplicated by peptides not derived from HCV NS3, whereas specific effects cannot.All recombinant proteins used in this study ( Fig. 1) were derived from the same infectious clone of HCV genotype 1a (4). NS3 spans amino acids 1027-1659 of the ϳ3,000-amino acid long polyprotein encoded by HCV. At the N terminus resides the pro...

The Functional Interaction of the Hepatitis C Virus Helicase Molecules Is Responsible for Unwinding Processivity

“…This rate is comparable or even faster than the reported nucleic acid unwinding rates of either the helicase domain or the full-length protease-helicase with or without the His tag (10,11,36). It was also shown recently that the DNA unwinding activity of the helicase domain with C-terminal or N-terminal His tag was similar (11).…”

“…The protease and helicase activities appear to be independent as these domains can be expressed separately in Escherichia coli while retaining their full activity (6 -9). Interestingly, NS3 helicase acts both on RNA and DNA and unwinds DNA better than RNA (10,11). This study is conducted with the bacterially expressed helicase domain of NS3 protein (NS3h) (12).…”

“…This study is conducted with the bacterially expressed helicase domain of NS3 protein (NS3h) (12). There is no consensus in the literature about the effect of the protease-NS4A on the helicase activity (10,11,(13)(14)(15)(16). The virus requires both helicase and protease functions, but it is not clear if the protease-NS4A activates or inhibits the helicase.…”

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

“…This view is difficult to envisage given the tight interaction of both domains observed in the crystal structure of full-length NS3 (Fig. 1B) (5) and by the finding that an isolated NS3 helicase fragment showed a slower kinetic of duplex RNA unwinding as compared with the full-length NS3 pro-tein (22). Moreover, NS4A appears to act as an RNA loading factor that greatly enhances productive RNA binding of a full-length NS3/4A complex (23) arguing for a cross-talk between protease and helicase domains.…”

“…The helicase domain is connected to the serineprotease domain via a flexible linker that may allow a rotation of both domains against each other and in this way a fine regulation of their respective enzymatic activities (Fig. 1B) (22,28). It will therefore be interesting to determine whether inhibitors of the protease also affect NS3 helicase activity.…”