NS2 proteases from hepatitis C virus and related hepaciviruses share composite active sites and previously unrecognized intrinsic proteolytic activities.

Over the recent years, several homologues with varying degrees of genetic relatedness to hepatitis C virus (HCV) have been identified in a wide range of mammalian species. HCV infectious life cycle relies on a first critical proteolytic event of its single polyprotein, which is carried out by nonstructural protein 2 (NS2) and allows replicase assembly and genome replication. In this study, we characterized and evaluated the conservation of the proteolytic mode of action and regulatory mechanisms of NS2 across HCV and animal hepaciviruses. We first demonstrated that NS2 from equine, bat, rodent, New and Old World primate hepaciviruses also are cysteine proteases. Using tagged viral protein precursors and catalytic triad mutants, NS2 of equine NPHV and simian GBV-B, which are the most closely and distantly related viruses to HCV, respectively, were shown to function, like HCV NS2 as dimeric proteases with two composite active sites. Consistent with the reported essential role for NS3 N-terminal domain (NS3N) as HCV NS2 protease cofactor via NS3N key hydrophobic surface patch, we showed by gain/loss of function mutagenesis studies that some heterologous hepacivirus NS3N may act as cofactors for HCV NS2 provided that HCV-like hydrophobic residues are conserved. Unprecedently, however, we also observed efficient intrinsic proteolytic activity of NS2 protease in the absence of NS3 moiety in the context of C-terminal tag fusions via flexible linkers both in transiently transfected cells for all hepaciviruses studied and in the context of HCV dicistronic full-length genomes. These findings suggest that NS3N acts as a regulatory rather than essential cofactor for hepacivirus NS2 protease. Overall, unique features of NS2 including enzymatic function as dimers with two composite active sites and additional NS3-independent proteolytic activity are conserved across hepaciviruses regardless of their genetic distances, highlighting their functional significance in hepacivirus life cycle

Hepacivirus NS2 autoprotease activity.

<p>(A) Schematic representation of the hepacivirus NS2-NS3_N-ST precursors. Precursors spanning NS2 and NS3 N-terminal domains (NS3_N) were expressed downstream of a heterologous signal peptide (sp) and C-terminally fused to Strep-tag (ST). NS3_N comprises two amino acid residues (H, D; thin solid lines) of putative or established NS3 protease catalytic triads, whereas the third residue (Ser) was mutated into Ala (SA, thin dotted line). NS2(wt)-NS3_N–ST precursors contain the native residues of the putative or established NS2 protease catalytic triads (H, E/D and C, thick solid lines). NS2(CA)-NS3_N-ST precursors bear an Ala substitution of the catalytic Cys residue (CA, thick dotted line) in NS2. (B) Proteins extracted from cells transfected with pCMV/NS2(wt)-NS3_N-ST or pCMV/NS2(CA)-NS3_N-ST DNAs, allowing expression of native or mutated NS2-NS3_N-ST precursors of the indicated viruses, respectively, were separated by SDS-PAGE and probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively.</p

NS2 N-terminal domain requirement for proteolytic activity.

<p>(A) Schematic representation of the hepacivirus ΔN(NS2)-NS3_N-ST truncated precursors, which are derived from NS2-NS3_N-ST polypeptides (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006863#ppat.1006863.g001" target="_blank">Fig 1</a> legend) and lack the hydrophobic N-terminal region of NS2. (B) Proteins extracted from cells transfected with pCMV/ΔN(NS2)(wt)-NS3_N-ST or pCMV/ΔN(NS2)(CA)-NS3_N-ST DNAs, allowing the transient expression of native (wt) or mutated (CA) ΔN(NS2)-NS3_N-ST truncated precursors (ΔN) of the indicated viruses, respectively, were separated by SDS-PAGE and immunodetected with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively. (C-D) HCV and GBV-B full-length (HCV, GBV-B) or truncated (ΔN HCV, ΔN GBV-B) precursors were expressed in a wheat germ cell-free expression system in the absence (C) or in the presence (D) of detergent MNG-3. Cell-free expression samples were affinity purified using Strep-Tactin beads, separated by SDS-PAGE and probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively.</p

Role and virus specificity of HCV NS3_N domain as NS2 protease cofactor.

<p>(A) Schematic representation of the chimeric NS2^HCV-NS3_N^hepaci-ST precursors. Precursors spanning NS2 from HCV-JFH1 and NS3_N from NPHV, BHV, GHV, RHV or GBV-B (^hepaci) were expressed downstream of a heterologous signal peptide (sp) and C-terminally fused to Strep-tag (ST). The sequence alignment of NS3 N-terminal sequences from the various hepaciviruses is depicted in the blown-up scheme with respect to HCV corresponding sequence, where identical residues are indicated by dots. (B) Extracts from cells transfected with pCMV/NS2^HCV(wt)-NS3_N^hepaci-ST or pCMV/NS2^HCV(CA)-NS3_N^hepaci-ST DNAs encoding NS2 protease with either native (wt) or mutated (CA) catalytic triad, respectively, were probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively. The decreasing overall sequence similarity of hepacivirus NS3_N with respect to HCV NS3_N is represented by a grey triangle.</p

HCV NS2 protease activity in the absence of NS3_N cofactor domain.

<p>(A) Schematic representation of (HAep)-NS2^HCV-(linker)-tag precursors. Precursors spanning HCV NS2 C-terminally fused to GFP, ST or V5 (tag), either directly or via a linker, were expressed downstream of a heterologous signal peptide (sp). Precursors used in panel D comprise a HA epitope (HAep, hatched box) introduced downstream of HCV NS2 N-terminal residue. HCV NS2 C-terminal residues, as well as GFP, ST or V5 N-terminal residues are displayed below the corresponding boxes. (B-D) Cells were transfected with pCMV/(HAep)-NS2^HCV-(linker)-tag DNAs that encode NS2 comprising either native (wt) or mutated (CA) catalytic triad and immediately fused to the linker indicated at the top of the panels or no linker (-) and GFP (B-C) or the indicated tag (D). Transfected cell extracts were probed with anti-GFP (B-C) or anti-HAep (D) antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively. Quantifications of cleavage rates (% cleaved products over total HAep-reactive precursors) were performed on 2–3 independent extracts subjected to infrared fluorescent immunoblot imaging and are plotted below representative blot images.</p

Dimerization of HCV, NPHV and GBV-B NS2 proteases.

<p>(A) Schematic representation of NS2 protease dimer formation upon co-expression of mutated precursors. The co-expression of NS2-NS3_N precursors (CA ST and HA V5 shown as examples) bearing an Ala substitution of either Cys (CA, red code) or His (HA, green code) catalytic residue and C-terminally fused to Strep-tag (ST, black code) or V5 tag (V5, blue code), respectively, may lead to the formation of NS2-NS3_N dimers. Homo- and hetero-dimerizations of NS2 protease, represented according to the three-dimensional crystallographic structure of NS2 catalytic domain (PDB accession number 2HD0) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006863#ppat.1006863.ref048" target="_blank">48</a>], result in the formation of native (wt), single-mutant (HA or CA) or double-mutant (HA and CA) NS2 active sites, as symbolized by scissors, one or two cross signs, respectively. (B-D) Cells were transfected or co-transfected with one (lanes 1 to 6) or two (lanes 7 to 10) pCMV DNAs, allowing the transient expression of NS2-NS3_N precursors from HCV-JFH1 (B), NPHV (C) or GBV-B (D). Uncleaved precursors and cleaved products were immunodetected with anti-ST or anti-V5 antibodies (upper and lower images, respectively in each panel) following SDS-PAGE separation of transfected cell extracts and are indicated by closed and open arrowheads, respectively.</p

Importance of hydrophobic surface residues in NS3_N for HCV NS2 protease activation by heterologous hepacivirus NS3_N.

<p>(A) Superposition of the backbone ribbon structures of GHV NS3_N (colored magenta) with HCV JFH1 NS3_N (colored cyan) homology models, established by using the crystal structures of HCV NS3 of genotype 1b as template (for details, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006863#sec011" target="_blank">Materials and Methods</a>). In the two images at the left (rotated by 90° one with respect to the other), the side-chain atoms of NS3 protease catalytic-triad residues (His 57, Asp 81, and Ser 139/140) are represented as blue spheres of the corresponding van der Waals radii for both models, and NS4A cofactors are represented in yellow. Residues I3, Y105, P115 and L127 in HCV (stick representation, colored green) are shown comparatively to the homologous residues F3, Y106, E116 and I128 (colored orange or red) in GHV. These surface residues are highlighted in the enlargement of NS3_N surface patch shown at the right. (B-E) HCV NS2-NS3_N-ST precursors with either native (wt) or mutated (CA) NS2 catalytic triads and HCV NS2-NS3_N-ST precursors containing the indicated substitutions at residues 105 and/or 115 of NS3_N were expressed in cells (B). Chimeric NS2^HCV-NS3_N^hepaci-ST precursors in which NS2 was derived from HCV and NS3_N was derived from NPHV (C), GHV (D), or GBV-B (E) and contained the indicated substitutions at residues 105, 115, and/or 127 were expressed in cells. Chimeric NS2^HCV-NS3_N^hepaci-ST controls harbored native (wt) or mutated (CA) HCV NS2 catalytic triads. (F-G) Hepacivirus NS2-NS3_N-ST precursors derived from NPHV (F), GHV (G), or GBV-B (H) with either native (wt) or mutated (CA) NS2 catalytic triads and precursors containing the indicated substitutions at residues 105 and/or 115/116 of NS3_N were expressed in cells. Transfected cell extracts were probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively. Dotted lines indicate where lanes originating from the same immunoblot image have been brought together. Quantifications of cleavage rates (% cleaved products over ST-reactive precursors + cleaved products) were performed on 2–5 independent extracts subjected to infrared fluorescent immunoblot imaging and are plotted below representative blot images. Stars above bars represent T test statistical analyses with respect to respective wt controls and are coded as follows: * p<0.01, ** p<0.001, *** p<0.0001, ns: non significant.</p

HCV NS2 protease substrate specificity in infected cells.

<p>(A) Schematic representation of the parental Jad-2EIL3 cDNA. Genomic sequences derived from a cell culture-adapted JFH1 variant (Jad) are represented by dark grey boxes. The heterologous EMCV IRES sequence followed by the Firefly luciferase (Luc), FMDV 2A peptide (2A) and ubiquitin (Ubi) coding sequences were inserted between NS2 and NS3 coding sequences, and are represented by light grey boxes. (B) Proteins extracted at 72h post-transfection from Huh7.5 cells transfected with Jad-2EIL3/HAep-NS2 or the indicated Jad-2EIL3/HAep-NS2-4GS-tag RNAs encoding NS2 protease with native (wt) or mutated (CA) catalytic triad were analyzed with antibodies specific for JFH1 NS2. Quantifications of cleavage efficiencies following infrared fluorescent imaging are derived from 2 independent experiments. (C) Genome replication. Relative intracellular luciferase activities were determined at 72 h post-transfection and expressed relatively to activities at 4 h post-transfection following transfection of Huh7.5 cells with the indicated RNAs encoding NS2 protease with native (wt, black bars) or mutated (CA, grey bars) catalytic triad. Controls include the parental Jad-2EIL3 RNA, the replication deficient Jad-2EIL3/GAA RNA and the assembly-deficient Jad-2EIL3/ΔEp7 RNA. NT: not tested (D) Infectious viral particle production was quantified by endpoint dilution titration of supernatants collected at 72h post-transfection and is expressed as log TCID50/mL titers. Means ± standard deviations of 3 independent transfections, each in duplicates are shown (C-D).</p

Phylogenetic analysis and multiple sequence alignments of NS2 and NS3 N-terminal domains of selected members of the <i>Hepacivirus</i> genus.

<p>NS2-NS3_N amino acid sequences of selected members of the <i>Hepacivirus</i> genus (for details, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006863#sec011" target="_blank">Materials and Methods</a>) were aligned with the T-coffee multiple sequence alignment program [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006863#ppat.1006863.ref074" target="_blank">74</a>] (A) A phylogenetic tree was constructed using the neighbor joining method under the Jones-Thornton-Taylor model of amino acid substitution implemented in the MEGA6 program [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006863#ppat.1006863.ref075" target="_blank">75</a>]. Bootstrap resampling from 2,000 replicates was performed in order to evaluate the reliability of grouping and significant values (>70%) are shown. The tree is drawn to scale with branch lengths proportional to the average number of amino acid substitutions per site, as indicated by the scale bar. Colored boxes cluster viruses according to their experimental (GBV-B) or natural (other viruses) hosts, as indicated above boxes. Viral strains considered in this study are highlighted in the respective species-coded colors. (B-C) The alignments of NS2 (B) and NS3_N (C) sequences of the indicated hepacivirus species representatives are shown with respect to the HCV JFH1 (2a) reference sequences with amino acids numbered according to their positions within respective proteins. Gaps between sequences are indicated by hyphens. To highlight the amino acid conservation at each position, residues identical to HCV JFH1 are indicated by dots. In the <i>Homology</i> line, identical, highly similar and similar residues across all sequences are symbolized by asterisks, colons and dots, respectively, according to Clustal W conventions. The local robustness of the sequence alignments is displayed in the <i>Reliability</i> line, according to a color code from low (blue) to high (red) confidence scores, as illustrated in the reliability scale. Residues comprising the HCV and GBV-B NS2 (B) and NS3 (C) protease catalytic triads and corresponding residues in other hepacivirus sequences are shown in boldface red type as indicated by the red arrowheads. The Cys and His residues of HCV NS3_N residues that coordinate Zn²⁺ and aligned residues in the other hepacivirus sequences are shown in boldface green type as indicated by the green stars. The HCV residues forming hydrophobic NS3 surface patch and aligned residues in the other hepacivirus sequences are shown in boldface orange type as indicated by the orange dots.</p

Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction

International audienceHepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE: Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts