NS2 proteins of GB virus B and hepatitis C virus share common protease activities and membrane topologies

Abstract

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis.

Importance: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

FIG 1

Comparison of the predicted GBV-B NS2 secondary structure with the experimentally defined HCV NS2 secondary structure. Sequences of NS2 proteins from GBV-B (top half of each block) and from the Con1 and JFH1 strains of HCV subtypes 1b and 2a, respectively (bottom half of each block) were aligned with Clustal W. The alignment is shown in two sequential blocks, and amino acids are numbered with respect to their positions within GBV-B and HCV NS2 proteins. Identical, highly similar, and similar residues at each position of GBV-B and HCV NS2 proteins are indicated with asterisks, colons, and dots, respectively (similarity), according to Clustal W conventions. Gaps between GBV-B and HCV NS2 sequences are indicated by hyphens. The predicted secondary structure (Pred. Struct.) is indicated in italics above the GBV-B amino acid sequence (c, coiled; e, extended; h, α-helix; t, turn; ?, undefined). Residues exhibiting a propensity to partition into the lipid bilayer according to the interfacial hydrophobicity plot calculated with MPex are underlined. Predicted TM segments (consensus TM) are indicated as a succession of T's above the secondary-structure predictions. See Materials and Methods for details concerning the algorithms used for these predictions. The HCV NS2 secondary structure (Sec. Struct.) is depicted below the HCV JFH1 sequence. It was established based on the NMR structure of selected synthetic peptides representing TM segments of HCV 1b-Con1 NS2 (31, 34) as well as the crystal structure of the HCV 1a-H77 NS2 C-terminal subdomain (38) and membrane association properties of selected segments of this subdomain (67). Solid and dotted boxes delineate hypothetical (GBV-B) or experimentally confirmed (HCV) TM and membrane-associated segments, respectively. Residues comprising the HCV NS2 protease catalytic triad are boxed in gray, and these conserved residues are also highlighted in the GBV-B NS2 sequence. Selected segments of GBV-B NS2 (S1 to S7) that were fused to GFP in this study are schematically represented by shaded boxes above the GBV-B sequence, with the positions of amino acid boundaries indicated at both extremities.

FIG 2

GBV-B NS2 autoprotease activity. Cells were transiently transfected with pCMV/GB-NS2–3pro-ST, pCMV/JFH1-NS2–3pro-ST, pCMV/GB-NS2(C177A)-3pro-ST, or pCMV/JFH1-NS2(C184A)-3pro-ST DNA, respectively, driving the expression of native (wt) polypeptide precursors (NS2-3pro) spanning NS2 and the protease domain of NS3 from GBV-B or HCV-JFH1 fused at their C termini to a twin Strep-tag (ST) or precursors bearing an Ala substitution of the Cys residue comprising the putative (GBV-B) (C177A) or established (HCV) (C184A) NS2 catalytic triad, as indicated above the gels. Uncleaved precursors and cleaved products were immunodetected with anti-ST antibodies following SDS-PAGE separation of transfected-cell extracts prepared at 40 h posttransfection and are indicated by closed and open arrowheads, respectively. Molecular mass markers are indicated at the left.

FIG 3

Fluorescence analysis of the membrane association properties of GBV-B NS2 segments fused to GFP. (A) Schematic representation and membrane association properties of GBV-B NS2 segments expressed as a fusion with GFP. The series of GBV-B NS2 segments expressed as a C-terminal fusion with a Gly-Ser (G-S) linker and GFP, as depicted at the top, are represented in the blow-up scheme by horizontal lines spanning the corresponding NS2 sequence, with amino acid boundaries (numbering refers to the amino acid position in NS2) indicated at both extremities. S1-GFP was expressed downstream of a heterologous signal peptide (sp) derived from the cellular protein CD5. Putative TM and membrane-associated (MA) segments are depicted as dark and light gray boxes, respectively, with amino acid boundaries indicated at the top. Membrane association (+) or a lack of membrane association (−) properties, determined primarily by immunofluorescence patterns in transiently transfected cells (panel D and data not shown), are indicated at the right of each segment. S1 to S7 (in blue) were studied further. (B to D) Subcellular localization of NS2 segments fused to GFP in transiently transfected cells. Cells were transiently transfected with plasmid DNAs ensuring the expression of GFP (pCMV/GFP) (B) or fusion proteins comprised of GFP C-terminally fused to aa 27 to 59 of HCV Con1 NS2 (pCMVNS227-59-GFP DNA) (C) or fused to GBV-B NS2 sp-S1 and S2 to S7, as indicated above the images (pCMV/SX-GFP) (D). Forty hours later, cells were fixed and stained for ER (p63) or Golgi (GM130) compartments, as indicated at the top left corner of the images, for indirect immunofluorescence analysis. Cell nuclei were counterstained with DAPI. Cells were observed by using an Observer Z1 inverted fluorescence microscope (Zeiss) in the ApoTome mode using a 40× objective. Representative images showing autofluorescent GFP (green), ER or Golgi compartments (red), and cell nuclei (blue) are superimposed. Bar, 20 μm.

FIG 4

Membrane association properties of GBV-B NS2 segments examined by flotation gradients. Discontinuous sucrose gradients were layered on top of lysates prepared at 40 h posttransfection from Cos-1 cells transfected with plasmid DNAs ensuring the expression of GFP (pCMV/GFP) or GFP downstream of the CD5 signal peptide (pCMV-sp-GFP) (A) or fusion proteins comprised of GFP C-terminally fused to aa 1 to 27 of HCV Con1 NS2 (pCMVNS21–27-GFP DNA) (B) or fused to the indicated segment (sp-S1 and S2 to S7) of GBV-B NS2 (pCMV/SX-GFP) (C to I). In panel D, extracts from S2-GFP-expressing cells were also treated with Triton X-100 (+ Triton X-100) (bottom gel) prior to loading below sucrose gradients. Seven fractions (fractions 1 to 7) were collected from the top of the density gradients following equilibrium ultracentrifugation, and the triangles at the bottom of the panels indicate the percent sucrose density gradient. The ER-resident p63 and GFP fusion protein contents of each fraction were determined following SDS-PAGE separation and immunodetection with the corresponding antibodies, as shown in the top and bottom gels, respectively. Cellular p63, GFP, GFP fused to the CD5 signal peptide (sp-GFP), GBV-B S1-GFP fused to the CD5 signal peptide (sp-S1-GFP), HCV fusion protein [(1-27)-GFP], GBV-B SX-GFP fusion proteins (S1-GFP to S7-GFP), as well as truncated (t) forms of these fusion proteins are identified at the right. Molecular weight markers (in thousands) are indicated at the left.

FIG 5

Glycosylation pattern of NS2-GFP fusion proteins bearing an N-glycosylation acceptor sequence. Protein extracts were prepared at 40 h posttransfection from cells transfected with plasmid DNAs encoding fusion proteins comprised of GFP C-terminally fused to aa 1 to 145 of HCV Con1 NS2 (A) or aa 1 to 141 of GBV-B NS2 (B), in which an Asn-Ser-Thr consensus acceptor sequence for N-glycosylation framed by two flexible hinges was introduced downstream of the N-terminal residue (N) or the amino acid position within the respective NS2 proteins indicated above the gels. A mutated glycosylation tag (mut) harboring Gln instead of Asn within the glycosylation acceptor site served as a control. Extracted proteins, either untreated (−) or subjected to deglycosylation by treatment with peptide-N-glycosidase F (PNGase F) (+), were separated by SDS-PAGE and identified following immunodetection with anti-GFP antibodies. Molecular weight markers (in thousands) are indicated at the left. Glycosylated proteins are marked with an asterisk. Dotted lines indicate where noncontiguous lanes that belong to the same immunoblot image have been brought together, and solid lines delineate different immunoblots.

FIG 6

Circular dichroism and NMR analyses of GBV-B NS2 synthetic peptides. (A to C) CD spectra and NMR data for GBV-B NS2(2–32), NS2(32–57), and NS2(113–137) synthetic peptides, respectively. Far-UV CD spectra (left) were recorded in water (H₂O) or complemented with either 50% 2,2,2-trifluoroethanol (TFE), 1% l-α-lysophosphatidyl choline (LPC), or the following detergents: 100 mM sodium dodecyl sulfate (SDS), 100 mM N-dodecyl-β-d-maltoside (DDM), or 100 mM dodecyl phosphocholine (DPC). Summaries of sequential (i, i + 1) and medium-range (i, i + 2 to i, i + 4) NOEs of GBV-B NS2 peptides in 50% TFE are shown in the right panels. Sequential NOEs allowing the assignment of proline residues are indicated in red. Asterisks indicate that the presence of an NOE cross peak was not confirmed because of overlapping resonances or the lack of H assignment. Intensities of NOEs are indicated by the height of the bars.

FIG 7

NMR structure models of synthetic peptides representing putative membrane-associated segments of GBV-B NS2. (A to C) Ribbon-and-stick models of representative NMR structures of GBV-B NS2(2–32) (A), NS2(32–57) (B), and NS2(113–137) (C) peptides. The amino acid sequences of the corresponding peptides are depicted at the top of each panel, and boxes indicate α-helix residues. Based on the NOE-derived interproton distances reported in Fig. 6, sets of 50 structures were calculated with X-plor, and final sets of 39, 36, and 28 low-energy structures that fully satisfied the experimental NMR data were retained for NS2(2–32), NS2(32–57), and NS2(113–137) peptides, respectively. Superimpositions of each set of structures (data not shown) highlight the most well-organized helical part for each peptide, i.e., segments spanning residues 13 to 28, 37 to 51, and 118 to 132, exhibiting RMSDs of 0.97, 0.63, and 0.71 Å for the backbone atoms (C′, C_α, and N) for NS2(2–32), NS2(32–57), and NS2(113–137) peptides, respectively. Representative NMR structures of each peptide (PDB accession numbers 2LZP [A], 2LZQ [B], and 2MKB [C]) are shown as ribbon-and-stick models, and the molecular surface of helices highlighting their hydrophilic and hydrophobic sides is shown in the middle and left views. The corresponding helix projections are shown on the right. Residues are color coded according to Wimley-White hydrophobicity scales (55), as follows: hydrophobic residues are gray; Phe and Trp are black; the polar residues Gly, Ala, Ser, Thr, Asn, and Gln are yellow; positively and negatively charged groups of basic and acidic residues are blue and red, respectively; and His is cyan. Figures were generated from structure coordinates by using VMD (http://www.ks.uiuc.edu/Research/vmd/) and rendered with POV-Ray (http://www.povray.org/).

FIG 8

Deduced model of GBV-B NS2 membrane topology. (A) In the model of the GBV-B NS2 membrane topology deduced from this study, TM segments 1 and 2 are shown in a ribbon-and-stick representation, according to representative NMR structures of the corresponding peptides. Residues are color coded according to their physicochemical properties, as described in the legend of Fig. 7. TM3, for which no NMR structure is available, is represented by an orange cylinder. The three TM segments are tentatively positioned as separate entities in the membrane, and the boundaries of TM α-helices are indicated on each side of the helices. Connecting sequences between TM segments are tentatively represented within membrane interfaces by dotted lines. The C-terminal cytosolic domain of GBV-B NS2 is represented by a three-dimensional homology model of one dimer subunit constructed by using the crystal structure of the HCV NS2 cytosolic domain as the template (PDB accession number 2HD0) (38) and the Swiss-Model automated protein structure homology modeling server (http://www.expasy.org/spdbv/). The resulting tentative model includes an α-helix spanning residues 116 to 135 (H2) with demonstrated membrane association properties, schematized as a dark pink cylinder within the plane of the membrane. (B) The HCV NS2 membrane topology model relies on the NMR structure of peptides representing the three N-terminal TM segments (31, 34) confirmed by the determination of the TM segment orientation with respect to the ER membrane (this study), the three-dimensional crystallographic structure of one dimer subunit of the C-terminal cytosolic domain (38), and the membrane association of this domain via α-helices H1 and H2 (67).

FIG 9

trans-complementation of the assembly-deficient Jad/S168E mutant containing a point mutation in the NS2 protease domain by NS2 proteins from HCV strains of various genotypes or from GBV-B. (A) Helper NS2 expression levels. Proteins were extracted at 48 h posttransfection from cells transfected in the absence of RNA (mock), transfected with cell culture-adapted Jad genomic RNA or with Jad/S168E genomic RNA encoding the S168E substitution within NS2, or cotransfected with Jad/S168E RNA and subgenomic V5-NS2 helper RNAs (in boldface type) expressing NS2 proteins from the indicated HCV strains (JFH1, J6, and N) or from GBV-B (GB), which were N-terminally tagged with a V5 epitope. Proteins were separated by SDS-PAGE and analyzed with antibodies specific for JFH1 core (top), V5 (middle), or JFH1 NS2 (bottom). Molecular weight markers (in thousands) are indicated at the left. Note that NS2 expressed by Jad/S168E genomic RNA (NS2^JFH1) is distinguishable from NS2 expressed by the subgenomic helper RNA that is fused to the V5 tag (V5-NS2^JFH1/J6), therefore migrating with a higher apparent molecular mass. Also note that NS2 antibodies recognize only HCV genotype 2a (JFH1 and J6) NS2 proteins. (B) Infectivity rescue. Huh-7.5 cells were cotransfected with either Jad (gray bars) or Jad/S168E (black bars) genomic RNA and the indicated V5-NS2 helper RNAs (HCV genotypes are shown in parentheses) or transfected with genomic RNAs in the absence of helper RNA (−). Release of infectious viral particles was determined at 48 h posttransfection by TCID₅₀ titration of transfected-cell supernatants in Huh-7.5 cells. Means and standard deviations of ≥3 independent transfections are shown.

FIG 10

trans-complementation of the Jad/S168E assembly-deficient mutant by HCV intra- or intergenotypic chimeric NS2 or by GBV-B/HCV chimeric NS2 with the native JFH1 protease domain. (A) NS2 expression from helper RNAs. Proteins were extracted at 48 h posttransfection from cells transfected in the absence of RNA (mock) or transfected with Jad or Jad/S168E genomic RNA or with the indicated subgenomic helper replicons (in boldface type) encoding E2–p7-NS2 from JFH1 (NS2^JFH1) or E2–p7-NS2 precursors in which chimeric NS2 proteins are comprised of N-terminal TM segments 1, 2, and 3 and membrane-associated segments [TM(123)-MA]; N-terminal TM segments 1, 2, and 3 [TM(123)]; or N-terminal TM segment 1 [TM(1)] from the J6 strain of HCV genotype 2a; the N strain of HCV genotype 1b, or GBV-B (GB) and the remaining NS2 sequences derived from JFH1 NS2 (see schematic representations at the bottom of panel C). Proteins were separated by SDS-PAGE and analyzed with antibodies specific for JFH1 NS2. Note that NS2 antibodies recognize the C-terminal protease domain of JFH1 NS2 and hence detect all chimeric NS2 proteins. (B) Core expression from genomic RNAs. Proteins extracted from cells transfected in the absence of RNA (mock), transfected with Jad or Jad/S168E genomic RNAs, or cotransfected with Jad/S168E RNA and the indicated helper RNAs (in boldface type) were separated by SDS-PAGE and analyzed with antibodies specific for core. (C) Infectivity rescue. Huh-7.5 cells were cotransfected with either parental Jad (gray bars) or assembly-deficient Jad/S168E (black bars) genomic RNAs and no helper RNA (−) or subgenomic helper replicons encoding E2–p7-NS2 precursors which comprise JFH1 NS2 or the indicated chimeric NS2s. These are described above for panel A and are schematically represented at the bottom with JFH1 and heterologous sequences depicted by gray and black boxes, respectively. Release of infectious viral particles was determined at 48 h posttransfection by TCID₅₀ titration of transfected-cell supernatants in Huh-7.5 cells. Means and standard deviations of ≥2 independent transfections are shown.

FIG 11

Multiple-sequence alignment of NS2 proteins from GBV-B, HCV, and new members of the Hepacivirus genus. (A) NS2 amino acid sequences of selected members of the Hepacivirus genus (for details, see Materials and Methods) were aligned with Clustal W, and a phylogenetic tree was constructed by neighbor joining with the MEGA6 program. Bootstraps were performed from 2,000 replicates, and values of >70% are shown. The bar indicates the average number of amino acid substitutions per site. The putative N- and C-terminal boundaries of NS2 proteins from BHV, GHV, NPHV, and RHV were based on predicted signal peptidase cleavage sites by using the SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/) and/or sequence homology with HCV and GBV-B. Colored circles cluster viruses according to their experimental hosts (GBV-B) or natural hosts (other viruses), as indicated at the surface of the circle. Viral strains considered for NS2 alignment (in panel B) are highlighted in the respective species-coded colors. (B) The sequences of the indicated six hepacivirus species representatives were aligned with the T-Coffee multiple-sequence alignment tool (74), using the corresponding Web server facility (http://tcoffee.vital-it.ch/). The alignment is shown in two sequential blocks, and amino acids are numbered with respect to their positions within NS2 proteins of GBV-B (top) and HCV (bottom). Gaps between NS2 sequences are indicated by hyphens. Identical, highly similar, and similar residues at each position of the six hepacivirus NS2 proteins are symbolized in the Reliability line by asterisks, colons, and dots, respectively, according to Clustal W conventions. This line also displays the local reliability of the sequence alignment according to a color code from low (blue) to high (red) reliability. Residues forming the HCV NS2 protease catalytic triad, which are identical (His and Cys) or highly conserved (Glu/Asp), are highlighted in cyan. The secondary-structure consensus (Sec.Struct.Cons) is indicated as helical (h) (blue), extended (e) (red), or undetermined (coil [c]) (orange). In the NS2 membrane binding domain (top block), this consensus was deduced from the experimentally determined peptide structures for GBV-B NS2 (this study) and HCV NS2 (31, 34) and secondary-structure predictions for GHV, BHV, RHV, and NPHV. The latter predictions were made by using the Web-based algorithms HNNC, MLRC, PHD, Predator, SOPM, and SIMPA96, available at the NPSA website (http://npsa-pbil.ibcp.fr). In the NS2 cytosolic domain (bottom block), the secondary-structure consensus was deduced from the crystal structure of the HCV NS2 cytosolic domain (PDB accession number 2HD0) (38) and the three-dimensional homology models for GBV-B, GHV, BHV, RHV, and NPHV NS2 proteins, constructed by using the HCV NS2 crystal structure as the template and the Swiss-Model automated protein structure homology modeling server (http://www.expasy.org/spdbv/) (75). For each viral sequence, amino acids predicted to belong to hydrophobic membrane segments by dense alignment surface (DAS) analysis (76) (http://www.sbc.su.se/∼miklos/DAS/) are shown in orange and red according to their DAS scores (1.7 and 2.2, respectively). Underlined residues correspond to predictions or experimental determinations of α-helices (blue underlining) and β-sheets (red underlining). Plain and dotted boxes delineate putative TM segments and membrane-associated α-helices (H), respectively, deduced from experimental studies of HCV NS2 (31, 34, 67) and GBV-B NS2 (this study).