BIS as a methodology to decrypt virus entry mechanisms.

<p>Schematic representation of the experimental approach employed in this study, from BIS computational analysis to the design and challenge of a mechanistic model of viral fusion. Following sequence analysis, matrix of E1E2 amino-acid coevolution were generated by BIS for different HCV genotypes. Plotting of matrix coevolution networks onto E2core structure unveiled a potential scenario of E1 and E2 rearrangements during HCV fusion, which involved the BL domain of E2. At the protein domain level, the construction of a soluble form of the BL and the conduction of several experimental assays supported such hypothesis. In parallel, at the amino acid level, the experimental validation of coevolution signals between specific residues of E1 and of the BL highlighted the critical role of E1-BL networks in regulating fusogenic rearrangements (and more generally, the critical role of coevolving networks between E1 and E2 C-terminal regions). Altogether, this approach allows us to propose a HCV fusion model where BL movements and E1 refolding are critical in the induction of E1E2 interdependent, fusogenic rearrangements. By being applicable to other viral proteins and viruses, such approach provides opportunities to uncover undescribed viral-mediated mechanisms and design innovative translational strategies for their inhibition.</p

BIS uncovers a coevolving signal between E1 and the Stem region that regulate HCV fusion.

<p>(<b>A</b>) Position of the three amino acid residues that differs between H77 (blue) and A40 (red) and are hypothesized to coevolve according to BIS prediction (gt1a cluster 5). The three H77 amino acids will be replaced by A40 residues individually or altogether to challenge BIS prediction. (<b>B</b>) Impact of the E1/Stem coevolution signal on HCV entry. Infectious titers of HCVpp viral particles harboring H77 (blue), A40 (red) and H77/A40 E1E2 chimera were determined. Two E1 H77 residues (S112, I117), a single H77 E2 residue (D462) or both (S112, I117, D462) were introduced into E1E2 A40. The different envelopes were incorporated at the surface of HCVpp, then used to infect Huh7.5. Infectious titers were quantified 72h post infection by flow cytometry (mean ± SD; n = 3). *<i>p</i><0.05, ns non-significant. (<b>C</b>) H77/A40 E1 and E2 chimera expression and incorporation onto HCVpp. Expression in transfected 293T cells (Cell lysates) and incorporation onto concentrated pseudoparticles (Viral Pellets) of E1 and E2 from the different H77/A40 chimera. Detection of E1 and E2 onto pseudoparticles harboring no envelope glycoproteins was used as negative control. MLV-Capsid (CA) was detected to control equivalent HCVpp production between chimera. (<b>D</b>). Impact of the E1/Stem coevolution signal on HCV fusion. LTRhiv-luciferase vector transduced 293T cells expressing the different E1E2 H77/A40 chimeric envelope glycoproteins were co-cultured with Tat-expressing Huh7.5 cells. Co-cultured cells were exposed to an acid shock (pH5, orange) or not (pH7, red) and luciferase activities were determined 72h post-exposure. Results are presented in relative light units (RLU) for each experimental condition (mean ± SD; n = 3). *<i>p</i><0.05, ***<i>p</i><0.001.</p

BIS analysis of dengue E coevolving networks during pre- and post-fusion steps.

<p>(<b>A</b>) Linear representation of Dengue E protein. Starting and ending residue positions of each E domain are indicated. E domains are annotated by distinct colors: DI, domain I (red); DII, domain II (yellow); DIII, domain III (blue); Tmd, transmembrane (black). (<b>B</b>) Organization and positions of the DENV E cluster 8 blocks (dark blue) on a mature E dimeric structure (PDB 1K4R). A linear representation of E is depicted and cluster block locations are indicated (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s004" target="_blank">S2 Table</a></b>). Cluster 8 coevolving residues located in areas where the two E monomers are in close contact are enlarged (green square). Structural proximity of two-coevolving cluster 8 internal loops located into two distinct Domain II (DII) sub-domains on the linear structure is also highlighted (red square). (<b>C</b>) Positions of E cluster 5 (red) and 10 (green) blocks on a mature tridimensional E monomer at a pre-fusion state (left, PDB 1K4R) and post-fusion state (right, PDB 1OK8). Stem and transmembrane domains are represented by a grey dotted line. A linear representation of E and cluster block locations are indicated for each cluster (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s004" target="_blank">S2 Table</a></b>). At the top of the panel, a schematic represents the current experimentally-validated fusion model of DENV, and how DENV E rearranges during this process. DENV E domains are colored into distinct colors (red, domain I; yellow, domain II; blue, domain III) as in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g001" target="_blank">Fig 1A</a></b>.</p

BIS analysis of dengue E-Pr coevolving residues.

<p>(<b>A</b>) Tridimensional representation of DENV Pr (Black, PDB 3C6R) and E (multi-color, PDB 1K4R). A linear representation of the PrM-E polyprotein is depicted below the protein structures. Starting and ending residue positions of each protein (Pr, M and E) and E domain are indicated. E domains are annotated by distinct colors: DI, domain I (red); DII, domain II (yellow); DIII, domain III (blue); Tmd, transmembrane (black). (<b>B</b>) Organization and positions of the PrM-E cluster 2 (orange), 7 (blue) and 9 (pink) on tridimensional representations of the DENV E and Pr proteins. Cluster 2 and 9 are depicted on a dimeric or trimeric E-Pr structure respectively at low pH condition (PDB 3C6R). Cluster 7 is depicted on a trimeric E-Pr structure at neutral pH condition (PDB 3XIY). Linear representation of the two proteins are also depicted on the top of each structure and cluster block location are indicated (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s003" target="_blank">S1 Table</a></b>). The close proximity between DENV E and Pr cluster 2 blocks is enlarged.</p

An E2 BLd-derived peptide inhibits HCV infection.

<p>(<b>A</b>) Putative BL movements that can modulate E2 rearrangements within E1E2 heterodimers. Rotation angles of the E2core structure are indicated (Reference: <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g003" target="_blank">Fig 3B</a></b>) and illustrated by a black cross harboring 4 color extremities. (<b>B</b>) Schematic representation of the E2 core structure and of the two hypothetical E2 conformational states prior (stretched) and following (packed) virus fusion when E1E2 are associated as heterodimer (color code and angle of E2 structures refer to <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g003" target="_blank">Fig 3B</a></b>). (<b>C</b>) Schematic representation of the expression construct encoding for the BLd-H77 peptide derived from the gt1a H77 E2 glycoprotein. SP, signal peptide; 6H, 6His-tag. (<b>D</b>) Detection of BLd-H77 in non-reducing condition following SDS-Page electrophoresis and coomassie blue staining. Molecular weight of the reference ladder are indicated on the left (kDa). (<b>E</b>) Detection of BLd-H77 peptide by Western Blot using a 6His-tag antibody. A 6His-tagged soluble E2 was used as a positive control. (<b>F</b>) Dose-dependent inhibition of HCVcc infection. HCVcc H77/JFH-1 or JC1 were pre-incubated with different doses of BLd-H77 (μg/ml) and used to infect Huh7.5 cells. Percentages of primary infection were calculated according to the viral titers of HCVcc particles incubated without BLd-H77 (mean ± SD; n = 3). (<b>G</b>) Kinetic of action of BLd-H77 on HCVcc infection. Huh7.5 cells were incubated 1h with BLd-H77 prior infection (35μg/ml) of HCVcc H77/JFH-1 (2), during the 4h infection (3) or following infection (secondary infection; 4). As control, cells were incubated at each step with equivalent volume of PBS (1). Percentages of primary infection were calculated according to viral titers of control conditions (mean ± SD; n = 3). Statistical significances <i>(</i>*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001) were determined for each experimental condition versus control condition (100%). (<b>H</b>) Dose-dependent inhibition of HCVpp harboring H77 envelope (HCVpp-H77) or control pseudoparticles harboring VSV-G (VSVGpp) envelope glycoprotein. Pseudoparticles were pre-incubated with different doses of BLd-H77 (μg/ml) or with PBS and used to infect Huh7.5 cells. Percentage of GFP positive cells was determined and expressed as percentages of infection, according to the viral titers of HCVpp incubated with PBS (mean ± SD; n = 3). (<b>I</b>) Kinetic of action of BLd-H77 on HCVpp infection. Huh7.5 cells were incubated 1h with BLd-H77 prior HCVpp H77 infection (50μg/ml) (2), during the 4h infection (3) or following infection (4). As control, cells were incubated at each step with equivalent volume of PBS (1). Percentages of infection according to viral titers of control conditions are reported (mean ± SD; n = 3). Statistical significances <i>(</i>**<i>p</i><0.01, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>J</b>) Dose-dependent inhibition of HCVcc JC1 virus infection of primary human hepatocytes (PHH). JC1 particles were pre-incubated with different doses of BLd-H77 (μg/ml) or with PBS and then used to infect PHH. PHH cell culture media were harvested four days post-infection and used to infect naïve Huh7.5 cells. Percentages of secondary infection are shown and calculated according to the viral titers of JC1 virus pre-incubated with PBS (mean ± SD; n = 3). (<b>K</b>) Dose-dependent inhibition of JC1-derived HCVpc (HCV primary cell culture-derived) of Huh7.5. HCVpc particles were pre-incubated with different doses of BLd-H77 (μg/ml) or with PBS and used to infect naïve Huh7.5 cells. Percentages of primary infection using HCVpc particles are shown and calculated according to the viral titers of JC1 virus pre-incubated with PBS (mean ± SD; n = 3). (<b>L</b>). Inhibition of HCV infection <i>in vivo</i> in humanized liver mice. JC1 infectious titers (FFU/ml) were obtained from two independent mice cohorts treated with 30μg (red, n = 3) or 150μg (blue, n = 2) of BLd-H77, or with PBS (black; cohort = 6) under a “prophylactic” protocol. BLd-H77 or PBS was injected one day prior virus infection with JC1 HCVcc particles and subsequent injections were performed at day 1, 7 and 14 post-infection. Sera were harvested 7 (Week 1), 14 (Week 2) and 21 days (Week 3) post-infection. HCV viral titers were determined through infection of Huh7.5 cells with mouse sera and quantification of FFU/ml. One non-engrafted liver mice and one non-infected mice were used as negative controls (green, n = 2). N.D., non-detected.</p

A dialog between E1 residues and the BL modulates virus entry and fusion.

<p>(<b>A</b>) Representation of the gt2 fusion cluster 5 (orange; dotted circle and linked by a bold line) as a putative mediator of E2 BL rearrangements and fusogenic conformational changes. Putative movements of the E2 BL are indicated by dotted double arrows. Rotation angles of the E2core structure and black cross are indicated as in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g003" target="_blank">Fig 3B</a></b>. (<b>B</b>) Regions of interest to study the role of the cluster 5 (orange) and design of the gt2 chimera. Three regions were defined as cluster 5 blocks. One is located on E1 (30 aa; Region 1) and two are located in E2 BLd (Region 2: E2 408–436, N terminal region; Region 3: E2 437–456, C terminal region). (<b>C-D</b>) A dialog between E1 and the BL domain regulates virus entry. Infectious titers of HCVpp (GFP i.u./ml, <b>C</b>) and HCVcc (FFU/ml, <b>D</b>) viral particles harboring J6 (black), 2b1 (white) and J6/2b1 E1E2 chimera. Swapped regions (1, 2 or 3) are represented by white (2b1) or black (J6) boxes inserted into J6 or 2b1 linear representations, respectively (bottom). Infectious titers were quantified 72h (HCVpp) or 4 days (HCVcc) post infection of Huh7.5. (mean ± SD; n = 4); *<i>p</i><0.05.; ns non-significant. (<b>E-F</b>) A dialog between E1 and the BL domain regulates membrane fusion. Cell-cell fusion induced by the different E1E2 chimera (as described in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g005" target="_blank">Fig 5J</a></b>). HANA (Influenza Hemagglutinin-Neuraminidase) envelope glycoproteins were used as positive control. Data are presented as relative light unit (RLU) at pH5 (<b>E</b>) or as percentage of fusion (<b>F</b>) where pH7 RLU is considered as 100% fusion rate for each chimera (mean ± SD; n = 3); *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 ns non-significant. For panel (<b>F</b>), statistical significances were determined for each experimental condition versus control condition (100%).</p

A transmembranous form of BLd-H77 inhibits HCV infection.

<p>(<b>A</b>) BLd acts onto viral particles. H77 HCVpp or H77/JFH-1 HCVcc particles were pre-incubated with BLd-H77 (50μg/ml or 35μg/ml, respectively) or with PBS. Prior to infection of Huh7.5 cells, HCV particles and BLd-H77 mixes were diluted (1/5; +) or not (-) with cell culture media resulting in a BLd-H77 concentrations inhibiting infectivity by less than 20% (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g004" target="_blank">Fig 4F</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g004" target="_blank">Fig 4H</a></b>). Viral titers of primary infection were determined 72h (HCVpp) or 4 days (HCVcc) post-infection and expressed as percentages of infection according to PBS control experiments (mean ± SD; n = 3). Statistical significances <i>(</i>*<i>p</i><0.05, ***<i>p</i><0.001) were determined for each experimental condition versus control condition (100%). (<b>B</b>) Schematic representation of the BLd-H77 transmembrane form (BLd-tm) and its possible mode of action. An IgG2 hinge region (Hinge) and the transmembrane region of the CD34 molecule (MSD) were added to the C-terminal of BLd-H77. SP, signal peptide. (<b>C</b>) A BLd-H77 transmembranous form inhibits HCVcc infection. H77/JFH-1 HCVcc were used to infect non-transduced Huh7.5, Huh7.5-C46 (C46) and Huh7.5-BLd-tm (BLd-tm). Four days post-infection, FFU titers were determined for each cell type and percentages of primary infection were normalized according to the viral titers determined following Huh7.5 infection (mean ± SD; n = 3). Statistical significances <i>(</i>***<i>p</i><0.001, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>D</b>). Propagation of HCVcc viral particles in Huh7.5-BLd-tm cell cultures. Huh7.5 and Huh7.5 BLd-tm cells were infected with HCVcc H77/JFH-1 at a m.o.i. of 0.1. At day, 1, 3 and 5 post-infection, cell culture supernatants were harvested and used to infect naïve Huh7.5 cells. Viral titers of secondary infection were determined by NS5A immunostaining four days post-infection (mean ± SD; n = 3). (<b>E</b>) BLd-tm inhibits cell entry. Non-transduced Huh7.5 and Huh7.5-BLd-tm were infected with HCVpp H77 and VSVpp. 72h post infection, amount of GFP positive cells were quantified. Percentage of infection of Huh7.5-BLd-tm is normalized for each type of particle on the percentage of infection of non-transduced Huh7.5 (mean ± SD; n = 3). Statistical significances <i>(</i>**<i>p</i><0.01, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>F</b>) Soluble E2 binding to Huh7.5-BLd-tm cells. Different doses of soluble E2 were mixed with different concentrations of Huh7.5 and Huh7.5-BLd-tm cells and E2 binding was quantified by flow cytometry. Results represent E2 ability to bind Huh7.5-BLd-tm cells for each condition relatively to the basal E2 ability to bind Huh7.5 cells (determined as 0% binding) for the same condition (mean ± SD; n = 3). *<i>p</i><0.05. (<b>G</b>) Interaction between BLd-H77 and sE2 detected by ELISA. Different amounts of mouse IgG isotype, AR3B and BLd-H77 were coated overnight into 96-well plates. Coated peptides and antibodies were then incubated with 10ng of soluble E2 (sE2) or not. After washing, soluble E2 was detected using the rat anti-E2 antibody 3/11 and an anti-rat HRP antibody. After measurement of the optical density (O.D.) at 450nm, relative E2 binding was determined by calculating the ratio of O.D. between condition with 10ng of sE2 and no sE2, for each coating condition (mean ± SD; n = 3). **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001, ns non-significant. (<b>H</b>) BLd-H77 does not affect HCVcc binding. JC1 HCVcc particles were pre-incubated with BLd-H77 (35 μg/ml), Heparine (250 μg/ml) or PBS and mixed with Huh7.5 cells for 2h at 4. After washing, amounts of cell-associated viral particles were determined by RT-qPCR (mean ± SD; n = 3). Data are shown as percentage of binding, according to binding of HCVcc particles in control condition (PBS). Statistical significances <i>(</i>*<i>p</i><0.05, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>I</b>) BLd-H77 can inhibit HCV entry following particle binding. HCVpp particles (HCVpp-H77) were incubated with Huh7.5 in presence of BLd-H77 (50μg/ml) or AR4A (25 μg/ml) during binding (1h at 4°C; 2), entry (4h at 37°C following binding; 3), or following entry (72h at 37°C following media change; 4). GFP levels were quantified 72h post infection. Huh7.5 infected in a similar manner but non-treated with BLd-H77 or AR4A were used as control (1). Percentages of infection were calculated based on viral titer obtained from control conditions. (mean ± SD; n = 3). Statistical significances <i>(</i>***<i>p</i><0.001, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>J</b>) Effect of BLd-H77 on cell-cell fusion. LTR-luciferase-transduced 293T cells expressing HCV-H77 E1E2 or VSVG glycoproteins were co-cultivated with Tat-expressing Huh7.5 cells. Following pre-incubation with PBS or with 50μg/ml of BLd-H77, co-cultured cells were exposed to an acid shock (pH5) or not (pH7) and luciferase activities were determined 72h post-exposure. Percentage of fusion of HCV and VSVG glycoproteins at pH5 between control (PBS) or BLd-H77 are indicated (mean ± SD; n = 3). Statistical significances <i>(**p</i><0.01, ***<i>p</i><0.001) were determined for each experimental condition versus control condition (100%). (<b>K</b>) Effect of BLd-H77 on virus-liposome fusion assays. H77 HCVpp particles were pre-incubated with different dose of BLd-H77 (50μg/ml, green; 100 μg/ml, blue; 150 μg/ml, red) or not (PBS, black), and mixed with R18-labelled liposomes. Dequenching of R18 was quantified following sample acidification (pH5). Data are represented as non-linear polynomial fitted curves for each experimental condition and display the evolution of the fusion rate (%) over time. Curves are representative of three independent experiments.</p

BIS analysis of HCV E1E2 coevolving residues.

<p>(<b>A</b>) Number of intra-E1 (red), intra-E2 (blue) and inter-E1E2 (purple) clusters detected by BIS following analysis of E1E2 sequences belonging to genotype 1 through 6. (<b>B</b>) Linear representation of HCV E1E2 glycoproteins and tridimensional representation of E2core structure (PDB 4MWF). E2 domains and their residue positions are indicated. E2core layers and domains are highlighted by distinct colors (Green, BL; Red, central β-sandwich; Blue, front layer; Dark blue, CD81 BinL/CD81 binding loop; Light grey, central β-sandwich–back layer linker; Black dotted line, VR2/Variable Region 2; Grey dotted line, Stem; Black rectangle, Tmd/Transmembrane). HVR1, Hyper Variable Region 1. Rotation angles of the E2core structure are indicated. Viewing angle of E2core is indicated by a black cross harboring 4 color points at each extremity. (<b>C-E</b>) HCV E1E2 gt1a (<b>C-D</b>) and gt2 (2a+2b, (<b>E</b>)) fusion clusters are plotted on a vertical linear representation of E1 combined to the tridimensional view of E2core (PDB 4MWF). Each cluster is composed of blocks harboring a similar color, according to <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s015" target="_blank">S3 Fig</a></b> (gt1a) or <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s018" target="_blank">S6 Fig</a></b> (gt2). Panel <b>C</b> shows E1E2 dialogs mediated by gt1a fusion cluster 5 and 7 and panel <b>D</b> underlines E1E2 dialogs mediated by gt1a fusion cluster 8, 10 and 12. Panel <b>E</b> shows E1E2 dialogs mediated by gt2 fusion cluster 10 and 13. The Stem region (Stem) is represented by a dotted line following the C-terminal part of the BL. The transmembrane domain (Tmd) is represented as a rectangle following the Stem region. Bold lines link E1 and E2 cluster blocks that coevolved. For each cluster, block positions in E1 (at the left of the linear structure) and E2 (below boxes whose color match the color of the corresponding cluster) are indicated. Rotation angles of the E2core structure are indicated and the viewing angle of E2core is indicated by a black cross (as in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g003" target="_blank">Fig 3B</a></b>).</p