Model of p7 role in assembly and envelopment of HCV particles.

<p>(<b>1</b>) HCV assembly takes place at the ER membrane close to HCV replication complexes-containing double membrane vesicles (DMV) and to cytosolic lipid droplets (LD) harboring core and NS5A at their surface. Upon translation of the HCV genome, E1E2p7NS2 complexes form and accumulate at the ER membrane. (<b>2</b>) p7, <i>via</i> its N-terminal extremity, regulates (thin grey arrow) the interaction of NS2 with NS5A. (<b>3</b>) This interaction may allow the release of E1E2 from E1E2p7NS2 complexes at the assembly site of nascent viral particles on the one hand and co-recruitment of core and RNA to E1E2 on the other hand. Subsequently, the E1E2-free p7NS2NS5A complex leaves whereas a new E1E2p7NS2 complex reaches the assembly area (dotted arrows). (<b>4</b>) The process is reiterated with p7 from incoming E1E2p7NS2 complexes regulating the encountering of NS2 and NS5A, which leads to the further release of E1E2 and core/RNA that accumulate at the assembly site. (<b>5</b>) The process is repeated until the formation of a viral particle that buds in the ER lumen. (<b>6</b>) The NS2NS5A complex, regulated by p7, may recruit ESCRT components to induce scission of the nascent particle until full envelopment and egress.</p

HCVcc mutants with modified p7 amino-terminus impair NS2 and NS5A association.

<p>Huh7.5 cells expressing RNAs from parental <i>vs</i>. Jc1 HAHALp7 mutant viruses expressed alone or with wild-type p7 were analyzed at 72h. (<b>A</b>) Confocal microscopy of Huh7.5 cells expressing Jc1 or Jc1 HAHALp7 viruses and stained for HCV core (red), NS5A (green) and NS2 (blue). (<b>B</b>) Co-localization analysis from (<b>A</b>), displaying the Pearson’s correlation coefficients and the Manders’ overlap coefficients, as indicated. (<b>C</b>) Confocal microscopy of Huh7.5 cells expressing Jc1 or Jc1 HAHALp7 viruses and stained for HCV NS2 (red), E2 (green) and NS5A (blue). (<b>D</b>) Co-localization analysis from (<b>C</b>), displaying the Pearson’s correlation coefficients and the Manders’ overlap coefficients, as indicated. (<b>E</b>) Cell lysates were incubated with protein A/G agarose beads after pre-treatment, or not (beads), with NS2 antibodies. Immuno-precipitated complexes were eluted and analyzed by quantitative western blot (see representative western blot to the left). The graphs show the levels of NS5A proteins co-immuno-precipitated by NS2 antibodies normalized to the amount of immuno-precipitated NS2 in Jc1 or Jc1 HAHALp7 virus-expressing cells. The values are displayed relative to association of NS5A to NS2 immuno-precipitated from Jc1 virus-expressing cells. Data represent mean values ± SEM. The numbers of experiments performed are indicated below the graphs.</p

p7 ATMI mutant viruses display increased E2 secretion and decreased RNA and core secretion.

<p>Huh7.5 cells were electroporated with RNAs from parental or Jc1 HAHALp7 mutant HCVcc viruses expressed alone or with wild-type p7 or HAHALp7. Analyses were performed at 72h post-electroporation. All data are normalized by percentage of HCV-positive virus producer cells obtained as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.g003" target="_blank">Fig 3A</a>. Results obtained with other p7 ATMI mutant viruses are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.s004" target="_blank">S4 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.s005" target="_blank">S5 Fig</a>. (<b>A</b>) Levels of secreted E2 determined by quantitative western blot following GNA lectin pull down of cell supernatants. (<b>B</b>) Levels of secreted core as determined by CMIA (Chemiluminescent Microparticle ImmunoAssay). (<b>C</b>) Levels of secreted HCV RNAs as determined by RT-qPCR. (<b>D</b>) Ratio of extracellular E2 to intracellular E2. (<b>E</b>) Ratio of extracellular E2 to extracellular core. (<b>F</b>) Ratio of extracellular E2 to extracellular HCV RNA. (<b>G</b>) Specific infectivity relative to E2 amounts. (<b>H</b>) Specific infectivity relative to core amounts. (<b>I</b>) Specific infectivity relative to RNA amounts. All values are displayed relative to infectivity and expression of E2, core or RNA values determined in the supernatants of Jc1 virus-electroporated cells (<b>A-I</b>). Data represent mean values ± SEM. The numbers of experiments performed are indicated below the graphs.</p

HCVcc mutants with modified p7 amino-terminus impair NS2 and NS5A association.

<p>Huh7.5 cells expressing RNAs from parental <i>vs</i>. Jc1 HAHALp7 mutant viruses expressed alone or with wild-type p7 were analyzed at 72h. (<b>A</b>) Confocal microscopy of Huh7.5 cells expressing Jc1 or Jc1 HAHALp7 viruses and stained for HCV core (red), NS5A (green) and NS2 (blue). (<b>B</b>) Co-localization analysis from (<b>A</b>), displaying the Pearson’s correlation coefficients and the Manders’ overlap coefficients, as indicated. (<b>C</b>) Confocal microscopy of Huh7.5 cells expressing Jc1 or Jc1 HAHALp7 viruses and stained for HCV NS2 (red), E2 (green) and NS5A (blue). (<b>D</b>) Co-localization analysis from (<b>C</b>), displaying the Pearson’s correlation coefficients and the Manders’ overlap coefficients, as indicated. (<b>E</b>) Cell lysates were incubated with protein A/G agarose beads after pre-treatment, or not (beads), with NS2 antibodies. Immuno-precipitated complexes were eluted and analyzed by quantitative western blot (see representative western blot to the left). The graphs show the levels of NS5A proteins co-immuno-precipitated by NS2 antibodies normalized to the amount of immuno-precipitated NS2 in Jc1 or Jc1 HAHALp7 virus-expressing cells. The values are displayed relative to association of NS5A to NS2 immuno-precipitated from Jc1 virus-expressing cells. Data represent mean values ± SEM. The numbers of experiments performed are indicated below the graphs.</p

Co-localization of p7 ATMI mutant and E2 with core protein in HCVcc infected cells.

<p>Confocal microscopy analysis of Huh7.5 cells infected with Jc1 HAHALp7 virus. At 72h post-infection, cells were fixed, permeabilized with either Triton X-100 or Digitonin, as indicated, and stained for HCV core (red), HAHALp7 (green) (<b>A</b>), E2 (green) (<b>B</b>) and nuclei (grey). The relative fluorescence intensity (arbitrary units) of each channel (<b>C</b>) was quantified by using ImageJ.</p

p7 ATMI mutant viruses do not alter E2, core, NS4B, NS5A and HCV RNA(+) clustering.

<p><b>(A)</b> Confocal microscopy of Huh7.5 cells infected with Jc1 or Jc1 HAHALp7 viruses. At 72h post-infection, cells were fixed and stained for HCV core (red), E2 (green), NS4B (blue, left panels), NS5A (blue, right panels) and nuclei (grey). <b>(B)</b> Quantification of size (left) and number (right) of co-localized core/E2/NS4B or core/E2/NS5A structures. (<b>C</b>) Confocal microscopy of Huh7.5 expressing Jc1 or Jc1 HAHALp7 viruses. At 72h post-infection, cells were fixed and stained for HCV core (green), NS4B (blue, left panels), NS5A (blue, right panels) and nuclei (grey). HCV RNA(+) were stained by FISH (red). <b>(D)</b> Quantification of percentage of core/NS4B or core/NS5A structures co-localizing with RNA(+) (left), of RNA(+) co-localizing with core (middle), or of RNA(+) co-localizing with NS4B/NS5A structures. Scale bars of panels and zooms from squared area represent 10μm and 2μm, respectively. For each condition, over 20 cells were quantified.</p

HCV p7 inhibits the cell secretory pathway.

<p><b>(A)</b> Ratio of extra- <i>vs</i>. intra-cellular HCV E2 glycoprotein determined by western blot analysis of cell lysates and of pellets of ultracentrifuged supernatants from Huh7.5 cells co-electroporated with Jc1 HCVcc RNAs and p7-expression (+ p7) or control (-) vectors, at 72h post-electroporation. (<b>B</b>) Intracellular expression of VSV-Gts-GFP fusion protein (VSV-Gts) and HA-tagged p7 assessed by flow cytometry, at 24h post-transfection. <b>(C)</b> Cell surface expression of VSV-Gts assessed by flow cytometry, using the 41A1 mAb directed against VSV-G ectodomain, after co-transfection of Huh7.5 cells with vectors encoding VSV-Gts and p7 (JFH1) expressed with the end of E2 (∆E2p7) or no signal peptide (noSPp7). Transfected cells were grown overnight at 40°C, which maintains VSV-Gts unfolded and results in its accumulation in the ER. Cells were then incubated for different periods of time (0h, 1h, 2h and/or 3h, as indicated) at 32°C, which allows restoration of its folding and thus, its secretion. The values represent the variations of the mean fluorescence intensity (delta MFI) of cell surface-expressed VSV-Gts relative to time 0h at 32°C. <b>(D)</b> Representative western blot analysis of cell lysates co-expressing VSV-Gts and p7, digested with endoH glycosidase. Cells were grown overnight at 40°C and lysed at different time points after incubation at 32°C. The endoH-resistant VSV-Gts species (arrows) indicates proteins that traffic to and beyond the Golgi apparatus. <b>(E)</b> Quantification of western blot from independent experiments performed as described in (<b>D</b>) after co-transfection of VSVG-ts with different p7 or with influenza H7N1 M2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.ref097" target="_blank">97</a>] protein-expressing constructs, as indicated. The values indicate the mean percentages of endoH-resistant VSV-Gts relative to total VSV-Gts for each time point. (<b>F</b>) p7 dose-dependence induction of endoH-resistance of VSV-Gts. <b>(G)</b> Representation of HCV E2 glycoprotein profile (expressed as percentage of total E2) in 3–40% iodixanol density gradients of subviral particles (SVP) produced after transduction of Huh7.5 cells with lentiviral vectors allowing the expression of either E1E2 or E1E2p7 proteins from J6 and/or JFH1 viruses. <b>(H)</b> Representative western blot of E2 detected with 3/11 antibodies in lysates and pellets of ultracentrifuged supernatants from cells producing SVPs (JFH1 strain). A range of dilutions of sE2 purified to homogeneity were run in parallel to the same western blot shown here. The intensity of E2 signals and the amounts of E2 are indicated. <b>(I)</b> Ratio of extra- <i>vs</i>. intra-cellular HCV E2 glycoprotein determined by quantification of western blots from independent experiments as described in (<b>H</b>). Data represent mean values ± SEM. The numbers of experiments performed are indicated below the graphs.</p

p7 ATMI mutant viruses increase the expression levels of HCV structural proteins.

<p>Huh7.5 cells were electroporated with RNAs from parental or Jc1 HAHALp7 mutant viruses expressed alone or with wild-type p7 or HAHALp7. Analyses were performed at 72h post-electroporation. Results obtained with other p7 ATMI mutant viruses are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.s003" target="_blank">S3 Fig</a>. <b>(A)</b> Flow cytometry analysis of HCVcc-expressing cells with NS5A antibody used to determine the proportion of HCV-positive virus producer cells. <b>(B)</b> Representative western blot analysis of cell lysates using antibodies against the indicated proteins. <b>(C)</b> Quantification of intracellular E2 levels. <b>(D)</b> Quantification of intracellular levels of core, NS2 and NS5A proteins. <b>(C-D)</b> Proteins were quantified and normalized after determining the proportion of HCV-positive virus producer cells (as determined in (<b>A</b>)) and the amounts of cellular actin. The values are displayed relative to expression of E2, core, NS2 and NS5A in Jc1 HCVcc RNA-electroporated cells. Data represent mean values ± SEM. The numbers of experiments performed are indicated below the graphs.</p

p7 ATMI mutant viruses induce secretion of viral particles with impaired envelopment.

<p>Huh7.5 cells were electroporated with RNAs from parental <i>vs</i>. Jc1 HAHALp7 or JFH1 HAHALp7 mutant viruses expressed alone or with wild-type p7 or HAHALp7, as indicated. Analyses were performed at 72h post-electroporation. <b>(A)</b> Levels of core and RNA following GNA pull down of cell supernatants, as determined by CMIA and RT-qPCR, respectively, and normalized by the amounts of immuno-precipitated E2. The values are displayed relative to association of core or RNAs to E2 pulled-down from the supernatants of Jc1 virus-electroporated cells. <b>(B)</b> Cell supernatants were digested with proteinase K (+ PK) with (+ TX) or without pre-treatment with Triton X-100 and the residual amounts of core (<i>i</i>.<i>e</i>., lipid membrane-protected core) were determined by CMIA. The values are displayed relative to non-treated conditions. <b>(C)</b> Analysis of density gradients of cell supernatants. Infectivity, core, RNA, and E2 were measured in each fraction and expressed as percentage of the sum of fractions. Examples of raw data of infectivity can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.s007" target="_blank">S7A–S7C Fig</a>. <b>(D)</b> Infectivity and core of the indicated viruses were measured in pooled fractions of densities of 1.08–1.15 g/ml and represented as data normalized by values obtained with parental viruses. The grey shaded area represents values below the sensitivity threshold of the experiments. Data represent mean values ± SEM.</p

HCVcc mutants with modified p7 amino-termini prevent infectivity.

<p><b>(A)</b> Schematic representation of JFH1 and Jc1 HCVcc mutants in E2-p7 junction. The first helix of p7 is shown in the light grey box. The insertions of residues are shown in dark grey and the substitutions are underlined. (<b>B-D</b>) Western blot analysis of Huh7.5 cells electroporated with RNAs from HCVcc viruses encoding wild-type or E2p7 cleavage mutants. <b>(B)</b> At 72h post-electroporation, cells were lysed and digested with endoH glycosidase before western blotting using 3/11 antibodies against E2. <b>(C)</b> The relative ratios of E2p7 precursors <i>vs</i>. free E2 species are indicated. <b>(D)</b> The HAHALp7 protein and the E2HAHALp7 (white arrows) and E2HAHALp7NS2 (black arrows) precursors were revealed using an HA tag antibody in lysates of cells electroporated with the indicated HCVcc constructs. <b>(E)</b> The extracellular (black) and intracellular (grey) infectivity levels for all mutants are represented. <b>(F)</b> The extracellular (black) and intracellular (grey) infectivity levels, normalized after determining the proportion of HCV-positive virus producer cells (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.g003" target="_blank">Fig 3A</a>), of JFH1 HAHALp7 or p7-T2 mutant viruses expressed alone or with wild-type p7 (+ p7) are represented relative to infectivity of parental JFH1 virus. <b>(G)</b> The extracellular (black) and intracellular (grey) infectivity levels, normalized by determining the proportion of HCV-positive virus producer cells, of Jc1 HAHALp7 mutant virus expressed alone or with wild-type p7 or HAHALp7, as indicated, are represented relative to infectivity of parental Jc1 virus. Data are displayed as means ± SEM. The numbers of experiments performed are indicated below the graphs.</p