New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay

Abstract

Background: The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a non-covalent heterodimer of the N-terminal, ∼180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attractive drug target.

Methodology/principal findings: Using a novel multiplex cleavage assay and over 2,660 peptide sequences derived from the polyprotein and from introducing mutations into the known NS3/4A cleavage sites, we obtained the first detailed fingerprint of NS3/4A cleavage preferences. Our data identified structural requirements illuminating the importance of both the short-range (P1-P1') and long-range (P6-P5) interactions in defining the NS3/4A substrate cleavage specificity. A newly observed feature of NS3/4A was a high frequency of either Asp or Glu at both P5 and P6 positions in a subset of the most efficient NS3/4A substrates. In turn, aberrations of this negatively charged sequence such as an insertion of a positively charged or hydrophobic residue between the negatively charged residues resulted in inefficient substrates. Because NS5B misincorporates bases at a high rate, HCV constantly mutates as it replicates. Our analysis revealed that mutations do not interfere with polyprotein processing in over 5,000 HCV isolates indicating a pivotal role of NS3/4A proteolysis in the virus life cycle.

Conclusions/significance: Our multiplex assay technology in light of the growing appreciation of the role of proteolytic processes in human health and disease will likely have widespread applications in the proteolysis research field and provide new therapeutic opportunities.

Figure 1. HCV polyprotein

(genotype 1a; GenBank accession P26664) with the NS3/4A cleavage sites. Non-structural (NS) proteins are shaded gray. Conventional NS3/4A cleavage sites in the NS3-NS4A, NS4A-NS4B, NS4B-NS5A and NS5A-NS5B junction regions are shown by solid arrows. The putative cleavage site (²⁴²⁹ALVTPC-AAEE²⁴³⁸) in the NS5A-NS5B junction is shown by a dashed arrow. The C–E1, E1–E2, E2–p7 and p7-NS2 junctions are cleaved by host cell signal peptidase. The NS2-NS3 junction is cleaved by NS2/3 auto-proteinase. C, core protein.

Figure 2. Results obtained after treatment of the 2,660 peptide substrate pool using NS3/4A.

(A) A scatter plot of peptide abundances in which Y-axis and X-axis represent the peptides following NS3/4A cleavage and untreated controls, respectively. The peptide abundances were determined by sequencing counts of cDNAs corresponding to these peptides. The red points represent peptides that exhibited a statistically significant change in abundance because of NS3/4A proteolysis. Z-score of 3 (p<0.0014) was set as a cutoff, which corresponds to a false discovery rate below 0.01. (B) A set of the 21 overlapping 8-mer peptides derived from the NS5A-NS5B junction region sequence (²⁴¹⁵EDVVCC↓SMSY²⁴²⁴). The Y-axis represents the Z-score for each peptide. The dotted red line represents Z-score = 3. Because the peptide sequences overlap, several adjacent peptides may contain sufficient recognition sequence to be cleaved. Peptide sequences are written vertically. Residue positions of the known NS3/4A cleavage site in the NS5A-NS5B junction region are highlighted (red boxes). The letter “A” at the start and at the end of each peptide sequence represents Ala residue from the flanking common regions at the N- and C-termini.

Figure 3. Frequency plot of the cleavage sequences of NS3/4A in a Weblogo format.

The size of the symbol indicates the frequency of the individual residue occurrence at individual substrate positions relative to the P1–P1′ scissile bond. The frequencies were calculated using over 2,660 peptide sequences derived from the polyprotein and from introducing mutations to the known NS3/4A cleavage sites.

Figure 4. Analysis of the NS3-NS4A, NS4A-NS4B, NS4B-NS5A and NS5A-NS5B junctions in the known HCV isolates.

The sequences that correspond to the P4–P4′ positions are shown. Substitutions relative to the HCV genotype 1a sequence (GeneBank Accession P26664) are in red. The frequency of the isolate and the Z-score of the peptide sequences are shown.

Figure 5. Structural modeling of NS3/4A with a protein substrate.

The ²⁴¹¹EANAEDVVCC↓SMSYSWTGAL²⁴³⁰ peptide that corresponds to the NS5A-NS5B junction region was used as a substrate in our modeling. (A) The structure of chymotrypsin – ecotin complex. This structure (PDB 1N8O) was used as a template for modeling the protein substrate in the NS3/4A structure (PDB 3LOX). Black, the catalytic triad. (B) The structure of the NS3 proteinase domain with the ²⁴¹¹EANAEDVVCC↓SMSYSWTGAL²⁴³⁰ modeled substrate. Black, the catalytic triad (His-57, Asp-81, Ser-139); yellow, Cys-159. (C) Close-up of the modeled NS3 structure. The negatively charged P5 Asp and P6 Glu of the substrate interact with the positively charged region of the NS3 proteinase that involves Arg-161, Lys-165 and Arg-123 (blue). (D) P5 Asp of the substrate interacts with Cys-159 of the NS3 proteinase. A dashed line shows a 3.8 Å distance between the carboxyl group of P5 Asp and the SH-group of Cys-159. (E) P6 Glu of the substrate interacts with Arg-123 of the NS3 proteinase. A dashed line shows a 2.6 Å distance between the carboxyl group of P6 Glu and the hydrogen of the guanidium group of Arg-123.