Serine Protease of Hepatitis C Virus Expressed in Insect Cells as the NS3/4A Complex

Abstract

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein into its constituent nonstructural proteins. The NS3/4A complex is thus an attractive target for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during expression, producing a noncovalent complex between HisNS3 and NS4A with a subnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatography. We removed the His tag by thrombin cleavage and then further purified the complex by gel filtration. The purified NS3/4A complex is active in a protease assay using a synthetic peptide substrate derived from the NS5A-NS5B junction, with kcat/K(m) of 3700 (+/- 600) M-1 s-1, an order of magnitude above those previously reported for NS3 expressed by other strategies. This high protease activity implies that the full-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activity is dependent on the aggregation state of the NS3/4A complex. The monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.

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

“…Therefore, His-NS3-4A was purified from insect cells infected with recombinant baculovirus containing an open reading frame encoding His-NS3-4A. The protein was expressed and purified using the procedure described for the same protein isolated from the similar HCV genotype 1a H strain (9). The purity of the recombinant HCV helicase proteins is shown in Fig.…”

“…HCV helicase was incubated at 37°C with a 32 P-labeled oligonucleotide with biotin at its 5Ј-end bound to streptavidin. When ATP is added to the reaction, streptavidin is displaced from the oligonucleotide leading to a shift in the electrophoretic mobility of the been observed by others using cross-linking (35), yeast twohybrid assays (17,39), and gel filtration chromatography (9,17). Although the vast majority of the proteins studied here migrate as monomers (or in the case of His-NS3-4A, a heterodimer), a small amount of the HCV proteins can be detected in the void volume of the columns, as was first reported by Sali et al (see Fig.…”

“…Baculovirus was plaque purified, amplified, and used for infection of Sf9 cells for protein purification. The protein was isolated from Sf9 cells using the protocol described by Sali et al (9). Recombinant proteins were monitored during the purification procedure by Western blotting using antibodies raised against NS3 and NS4A.…”

“…To analyze the helicase activities of the full-length protein, NS3 and NS4A were coexpressed as a single polypeptide that processes itself (9). The truncated helicase proteins lacking protease function (Fig.…”

The Hepatitis C Virus NS3 Protein: A Model RNA Helicase and Potential Drug Target

“…NS4A could hold the protein in a conformation so that the RNA binding cleft between the protease and helicase remains intact, explaining why some investigators find that a NS3-NS4A complex unwinds RNA better than NS3 alone. For example, Pang et al (Pang et al, 2002) compared the activities of a NS3-NS4A complex expressed in insect cells (Sali et al, 1998) with a His-tagged, full-length NS3 protein expressed and purified from E. coli, and found that the NS3-NS4A complex requires less time to form a functional complex on RNA. Based on the structure of the NS3-NS4A complex (Fig.…”