Hepatitis C Virus NS3 ATPases/Helicases from Different Genotypes Exhibit Variations in Enzymatic Properties

Abstract

The NS3 ATPase/helicase was isolated and characterized from three different infectious clones of hepatitis C virus (HCV). One helicase was from a genotype that normally responds to therapy (Hel-2a), and the other two were from more resistant genotypes, 1a (Hel-1a) and 1b (Hel-1b). Although the differences among these helicases are generally minor, all three enzymes have distinct properties. Hel-1a is less selective for nucleoside triphosphates, Hel-1b hydrolyzes nucleoside triphosphates less rapidly, and Hel-2a unwinds DNA more rapidly and binds DNA more tightly than the other two enzymes. Unlike related proteins, different nucleic acid sequences stimulate ATP hydrolysis by HCV helicase at different maximum rates and with different apparent efficiencies. This nucleic acid stimulation profile is conserved among the enzymes, but it does not result entirely from differential DNA-binding affinities. Although the amino acid sequences of the three proteins differ by up to 15%, one variant amino acid that is critical for helicase action was identified. NS3 residue 450 is a threonine in Hel-1a and Hel-1b and is an isoleucine in Hel-2a. A mutant Hel-1a with an isoleucine substituted for threonine 450 unwinds DNA more rapidly and binds DNA more tightly than the parent protein.The hepatitis C virus (HCV) evolves so rapidly that, in individual patients infected with certain genotypes, HCV exists as a heterogeneous population of quasispecies. Both viral genotype and quasispecies diversity influence disease severity and treatment response (reviewed in references 9 and 45). Patients infected with genotype 1 often do not respond to antiviral therapy, whereas patients with other genotypes respond more favorably (25). The kinetics of viral load decreases after the initiation of interferon therapy suggested a high freevirion clearance rate, a low rate of virus production in infected cells, and a high rate of infected-cell death in patients infected with genotype 2 relative to those in patients infected with genotype 1 (28). Strains that more rapidly evolve into diverse quasispecies more readily evade the host immune system, leading to chronic hepatitis (10), and respond less well to therapeutic intervention (11). Exactly how HCV genetic variation leads to these clinically important viral phenotypes is still largely a mystery, however. This comparative study was therefore initiated to define the impact of genetic variation on the activity of one of the best characterized HCV proteins, the NS3 helicase.HCV is a positive-sense, single-stranded RNA (ssRNA), and as such, when it enters a cell, HCV genomic RNA is translated into a peptide over 3,000 amino acids long from a single open reading frame. Both the host and viral proteases process the HCV polyprotein into three structural proteins (core, E1, and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Most studies dealing with the impact of genetic variation on the HCV life cycle have focused on linking heterogeneity in NS5A (7) and E2 (38) with the tre...

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

“…Cloning, expression, and purification of Hel-His (which was previously designated Hel-1a) have been described (15). A detailed enzymatic characterization of Hel-1a was reported recently (16).…”

“…250 nM trap DNA was also added before electrophoresis to prevent reannealing. Products were analyzed by nondenaturing PAGE as described previously (15). In some assays, excess trap DNA, which consisted of the unlabeled shorter oligonucleotide, was added when the reactions were initiated.…”

“…After cells had grown to mid log phase in Luria-Bertani broth, helicase expression was induced with 1 mM isopropyl-␤-D-thiogalactopyranoside. Cells were harvested and lysed by two passes through a French pressure cell, and the clarified soluble extract was fractionated as described previously for Hel-His (15).…”

“…His-Hel-His-To generate a HCV helicase fragment with both Cterminal and N-terminal His tags, DNA encoding the helicase was excised from plasmid p24Hel-1a (15), which encodes Hel-His, and inserted into the vector pET33a (Novagen, Madison, WI). Plasmid p24Hel-1a was digested with NheI and BamHI.…”

“…The truncated helicase proteins lacking protease function (Fig. 1A) were based on those previously analyzed and include versions with N-terminal (10 -12) and C-terminal (7,(13)(14)(15)(16) polyhistidine tags (His tags) and a version in which the protease is replaced with a glutathione Stransferase (GST) protein (17). The presence of an intact protease domain dramatically enhances the ability of the protein to unwind RNA.…”