The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target

Abstract

The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled byATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been proposed to explain how this conformational change leads to protein movement and RNA unwinding, but no model presently explains all existing experimental data. Compounds recently reported to inhibit HCV helicase, which include numerous small molecules, RNA aptamers and antibodies, will be useful for elucidating the role of a helicase in positive-sense single-stranded RNA virus replication and might serve as templates for the design of novel antiviral drugs.

Fig. 1

HCV Helicase Structures. (A). PDB file 1A1V, showing the HCV helicase with a bound DNA oligonucleotide and sulfate ion (Kim et al., 1998). The N-terminal RecA-like domain (domain 1) is colored yellow, the C-terminal RecA-like domain (domain 2) is purple, and domain 3 is pink. DNA and a sulfate ion (which occupies the ATP binding site) are depicted as spheres. (B) An electrostatic surface of the protein in 1A1V calculated without the DNA using the program APBS (Baker et al., 2001). Note the DNA is held in a negatively-charged pocket. (C) A full-length NS3 complex with the central portion of NS4A covalently tethered to the NS3 N-terminus (Howe et al., 1999), as seen in PDB file 1CU1 (Yao et al., 1999). Helicase domains are colored as in panel A with the protease colored green and NS4A blue. The protein is rotated about 90° relative to panel A. (D) An electrostatic surface of the protein as viewed in panel C. Note that the positively-charged cleft surrounding the protease, which could provide additional RNA binding sites. (E) Comparison of HCV helicase in the closed conformation (PDB file 1HEI (Yao et al., 1997)) and the open conformation (PDB file 8OHM (Cho et al., 1998)). Proteins are superimposed along domains 1 and 3. (F) Two NS3 helicase fragments (red, blue) bound to the same oligonucleotide (Mackintosh et al., 2006). Subunit 1 (red) of the helicase: DNA complex (PDB file 2F55) was aligned with the backbone the helicase portion of 1CU1. The surface of 1CU1, which includes the helicase and protease domains, is shown as transparent amber surface. All structures were rendered using the program Pymol (DeLano Scientific LLC, San Francisco, CA).

Fig. 2

HCV NS3 sequence conservation. A sequence logo (Schneider and Stephens, 1990) of an NS3 sequence alignment is overlaid on a cartoon of the HCV helicase. Each residue of NS3 is depicted as a stack of letters, the height of which correlates with how well it is conserved in 138 NS3 sequences deposited in the HCV database (http://hcv.lanl.gov/). The height of the letters in each stack correlates with how frequently that amino acid occurs at that position. Conserved superfamily 2 helicase motifs and other key residues are noted and highlighted with bold type.

Fig. 3

Key residues in HCV helicase. (A) HCV helicase residues likely involved in modulating ATP binding and hydrolysis. The approximate position of ATP bound to HCV helicase is revealed by a structural alignment of HCV helicase (PDB file 1A1V) (Kim et al., 1998) with the SF2 helicase RecQ bound to ATPγS (PDB file 1OYY) (Bernstein et al., 2003). (B) Key residues contacting the oligonucleotide bound to HCV helicase in PDB file 1A1V (Kim et al., 1998).

Fig. 4

Two possible mechanisms for HCV helicase translocation on RNA. (A) The Brownian motor model (Levin et al., 2005). In the absence of ATP, HCV helicase is confined in a single location on an asymmetrical path of RNA. When ATP binds, binding releases the protein from RNA, allowing random movement (Brownian motion) to transport the helicase either in a 5’ or 3’ direction. Because the path is asymmetrical, molecules moving in the 3’ direction will return to their original position, whereas molecules moving in the 5’ direction will change positions. Net movement will be in a 5’ direction. (B) The propulsion-by-repulsion model (Frick et al., 2004a; Lam et al., 2004). ATP binding rotates domain 2 so that a positively charged Arg-clamp (Lam et al., 2003a) moves the RNA so that it clears Trp⁵⁰¹, which is holding the RNA in a negatively charged cleft. When ATP is bound, the protein repels RNA past Trp⁵⁰¹ so that the protein moves in a 5’ direction until ATP is hydroiyzed and the protein returns to its original conformation.