Density profiles of HCV particles derived from Con1/wt, Con1/K1846T, JFH-1 and patient serum.

<p>(A) Approximately 22 ml of filtered cell culture fluid harvested 24 h after transfection of Huh-7 cells with Con1/wt RNA were concentrated via ultracentrifugation over a 60% iodixanol cushion. In case of JFH-1 approximately 26 ml supernatant harvested 96 h post transfection and concentrated in the same manner were used. The concentrates were overlaid with a linear iodixanol gradient (0%–60%) and spun for 20 h at 110,000 g at 4°C. In a similar experiment, 0.5 ml of high titer patient serum was resolved in an iodixanol density gradient. Twelve fractions à 1ml were harvested from the top, and the amount of HCV core protein contained in each fraction was determined by Trak C ELISA, respectively. HCV core content per fraction is plotted against the density of the respective fraction. (B) Result of an analogous experiment but using culture supernatant of Huh-7 cells after transfection with Con1/wt or Con1/K1846T.</p

Impact of REMs on HCV particle release.

<p>(A) Replication of subgenomic luciferase replicons in transfected Huh-7 cells. The replication deficient replicon D318N served as negative control. Values refer to luciferase activities determined 48 h post transfection of Huh-7 cells after normalization for transfection efficiency determined 4 h post transfection. Data are taken from reference <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000475#ppat.1000475-Lohmann3" target="_blank">[17]</a>. (B) Huh-7 cells were transfected with the Con1/wt genome or Con1 genomes carrying REMs specified in the bottom of the graph. The amount of core protein released into the supernatant 24 h after transfection was determined by using core ELISA and is expressed relative to the quantity of core measured for Con1/wt. Mean values of between 2 and 11 independent repetitions (depending on the construct) including the standard deviation are given. (C) Impact of REMs in NS4B and NS3 on RNA replication as determined by intracellular accumulation of core protein (left panel). The kinetic of release of core protein into the supernatant of Huh-7 cells after transfection with the Con1/wt or the Con1/K1846T or the Con1/NS3+K1846T genome is shown in the middle panel. The right panel shows the percentage of intracellular core that is released into the culture supernatant of Huh-7 cells at various time points after transfection with each of the 3 genomes. A representative example of two independent repetitions is shown.</p

Transient replication of HCV Con1-derived constructs in Huh-7 cells and release of core protein from transfected cells.

<p>(A) Schematic representation of transfected RNA genomes. The polyprotein is indicated by an open box, the individual functional proteins are separated by vertical lines. Non-translated regions are depicted as shaded bars, REMs and the position of the mutation destroying the active site of the NS5B RdRp (D318N) are specified above the respective positions in the coding region. (B) Transient RNA replication of full length Con1-derived genomes. Ten µg of <i>in vitro</i> transcribed RNA of the constructs specified in the top were transfected into Huh-7 cells that were harvested at given time points. Total cellular RNA was prepared and HCV RNA and beta-actin RNA were detected by Northern hybridization. (C) The amount of HCV RNA was determined by phospho imaging and is expressed relative to the input determined 4 h post transfection. Values were normalized for equal RNA loading as determined with the beta-actin specific signal. (D) Time course of accumulation of HCV core in cell culture supernatant of transfected Huh7 cells. Cells were transfected and seeded as described in (A). At given time points, culture medium was harvested, filtered through 0.45 µm pore-size filters, and analysed for core protein by ELISA. Duplicate measurements, mean value of duplicates and standard errors of the means are given. (E) Efficiency of core release from cells transfected with Con1/wt or the adapted genome Con1/NS3+S2197P <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000475#ppat.1000475-Bukh1" target="_blank">[27]</a>. Amounts of core protein accumulated intracellularly or in cell culture medium were determined by ELISA and used to calculate the percentage of intracellular core protein released into the supernatant of transfected cells for each given time point. Mean values of two independent electroporations including standard errors of the means are shown.</p

<i>In vitro</i> infectivity of Con1/wt particles released from transfected Huh7.5 cells.

<p>(A) Enhancement of HCV RNA replication by kinase inhibitor H479. Subgenomic Con1 luciferase replicons were transfected into Huh7.5 cells that were seeded into medium containing H479 at concentrations specified in the right. Cell lysates were prepared at 4 h and 48 h after transfection and luciferase activities were determined. The replication defective replicon Con1/D318N served as negative control. Cells treated with DMSO only were used as reference. For each construct, values were normalized to the luciferase activity of the respective DMSO control in order to determine the fold induction or reduction of replication. Data (mean±S.D.; n = 3) were analyzed using two-way ANOVA test. (B) Experimental approach used to detect <i>in vitro</i> infectivity of Con1 virus. (C) Immunofluorescence analysis of Huh7.5 cells 72 h after inoculation with supernatant from cells transfected with the Con1/wt genome (upper panels) or mock transfected cells (lower panels). Cells were treated either with DMSO only (Mock; left panels), or with H479 (middle panels) or with H479 and ConcanamycinA (right panels) as specified in panel (B). Cells were fixed 72 h after inoculation and NS5A was detected by immunofluorescence microscopy. (D) Detection of NS3 and NS5A expression in Huh7.5 cells inoculated with cell-free concentrated supernatant containing Con1/K1846T particles. Cells were fixed 48 h after inoculation and processed for indirect immunofluorescence. Nuclei were counterstained with DAPI.</p

<i>In vivo</i> infectivity of Con1/wt, Con1/K1846T and Con1/NS3+K1846T genomes in uPA-SCID mice.

<p>Huh7-Lunet cells were transfected with either of these constructs, supernatants were collected 12 and 24 h post transfection, pooled for each construct and used for virus purification and concentration as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000475#s4" target="_blank">Materials and Methods</a>. Two mice were each inoculated with 2×10⁸ IU HCV RNA per mouse and construct (100 µl inoculum size) and viral RNA loads in sera were determined at the indicated time points after inoculation by qRT-PCR. In case of Con1/K1846T inoculated mice, one died at week 2 (not shown) and the second shortly after week 6. While sera of Con1/wt and Con1/K1846T inoculated mice contained high viral loads already in the first blood sample, Con1/NS3+K1846T-inoculated mice remained HCV RNA negative throughout the 10 weeks observation period.</p

Release of HCV core into the supernatant of transfected cells depends on the expression of functional glycoproteins.

<p>Ten µg of <i>in vitro</i> transcribed RNA of the constructs specified below each bar were transfected into Huh-7 cells and 24 h later culture medium was harvested and filtered, whereas cells were lysed with 1% Triton X-100 in PBS. The total amount of HCV core in the cell lysate (A) and culture supernatant (B) was determined by ELISA. (C) Relative core release expressed as the fraction (in %) of total intracellular core protein that is released into the culture fluid. Mean values of two independent electroporations including the standard error of the means are given.</p

Hypothetical models describing the formation of double membrane vesicles and their possible role in viral RNA replication.

<p>(A) By analogy to flaviviruses <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003056#ppat.1003056-Welsch1" target="_blank">[9]</a> HCV proteins induce invaginations of the ER membrane. Extensive invagination leads to a local ‘shrinking’ of the ER lumen. This model assumes that enzymatically active HCV replicase (green dots) reside in the lumen of the invagination and remain active as long as the vesicle is linked to the cytosol. Upon closure of the DMV, the replicase would become inactive (grey dots). Alternatively, closed DMVs might be connected to the cytosol via proteinaceous channels. (B) HCV proteins might induce tubulation of ER membranes that undergo secondary invagination and thus double membrane wrapping. These DMVs could initially be open to the cytosol, but might close off as replication/infection progresses. The resulting DMV might stay connected to the ER via a stalk or be released as a ‘free’ DMV (left or right drawing, respectively). (C) Induction of DMVs follows the same pathway as described for panel B, but the viral replicase remains on their cytosolic surface as discussed e.g. for the poliovirus <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003056#ppat.1003056-Bienz1" target="_blank">[28]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003056#ppat.1003056-Egger2" target="_blank">[29]</a>. (D) HCV RNA replication might occur on SMVs in close proximity of DMVs. In this case, DMVs might be an epiphenomenon or serve some other purpose for the HCV replication cycle. For each model, structures identified in the 3D reconstructions are shown next to or below the corresponding schematic drawing. For further details see text.</p

Correlation between HCV RNA replication and appearance of double membrane vesicles.

<p>(A) Time course of spread of HCV infection in Huh7.5 cells infected with 100 TCID₅₀/cell of Jc1. Infected cells were detected by immunofluorescence using an NS5A-specific antiserum (upper panels). The graph shown below represents the result of counting ∼200 cells for each time point to determine the percentage of infected cells. Scale bars represent 50 µm. (B) Time course of accumulation of intracellular HCV RNA in infected Huh7.5 cells. The graph shows the result of two independent experiments (3 replicas each). Whiskers indicate the minimum and maximum values. (C) Colocalization of dsRNA and NS5A in cells infected with Jc1 (10 TCID₅₀/cell). Cells were fixed at time points specified in the left of each panel row and NS5A and dsRNA were detected by indirect immunofluorescence microscopy. DNA was stained with DAPI (blue). Boxed areas in the left panels indicate areas that are shown as enlargements in the corresponding right panels. The quantification of the degree of colocalization (Pearson's correlation coefficient) is given in the enlarged pictures. Scale bars represent 10 µm and 2 µm (left and right panels, respectively). (D) Time course of accumulation of DMVs and MMVs. For each time point, 10 cellular profiles were counted. The Mann-Whitney (non-parametric) test was applied to determine statistical significance. Error bars refer to the standard deviation. Note the striking correlation between the increase of intracellular HCV RNA and DMV number between 16 and 24 hpi.</p

3D architecture of membrane rearrangements induced 16 h after HCV infection.

<p>(A) Huh7.5 cells were infected with 100 TCID₅₀/cell of Jc1, fixed 16 h later and after HPF-FS processed for ET as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003056#s4" target="_blank">materials and methods</a>. Left: slice of a dual axis tomogram showing the various membrane alterations. Right: 3D reconstruction of the complete tomogram. Note the high number of DMVs. Panels B, C and D are part of the tomogram displayed in panel A and their position in panel A is highlighted by either a yellow dashed square (B and C) or by a star (D). In the 3D models shown on the right, the ER is depicted in dark brown, the inner membrane of DMVs and DMTs in yellowish brown and their outer membrane in semi-transparent light brown. Single membrane vesicles are colored in pink, intermediate filaments in dark blue and the Golgi apparatus in green. (B) Left: serial single slices through the same tomogram shown in panel (A) displaying a connection between the outer membrane of a DMV and the ER membrane (black arrows). Right: 3D surface model showing the membrane connection. (C) Left: serial single slices through the same tomogram illustrating a lasso-like structure of a DMV that after rendering reveals a pore-like opening that connects the interior of the DMV with the cytosol. The position of this opening in the 2D slice is marked with a black arrow. Right: 3D view of this DMV showing the ‘pore’. (D) Left: serial single slices through the same tomogram showing a DMV with a large inter-membrane space between its inner and outer membranes. Right: 3D view of this DMV. Scale bars represent 100 nm. This tomogram is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003056#ppat.1003056.s006" target="_blank">movie S1</a>.</p

Immuno-EM localization of NS5A and NS3 in HCV-infected cells.

<p>Huh7.5 cells were infected with 100 TCID₅₀/cell of Jc1 and fixed 16 h post-infection. (A) Cells shown in panels b, c and e to g were post-fixed with 0.1% OsO₄ providing best membrane preservation, especially in case of DMVs (f and g) and LDs (e). Both NS5A and NS3 localize predominantly to the ER (b and d) and 50–70 nm diameter single-membrane vesicles (SMVs; subpanel a, c and d). NS5A also localizes to lipid droplets (e). (B) Amount of gold particles per µm² in Jc1-infected versus mock-infected cells after immunolabeling with NS3- and NS5A-specific primary antibodies. Note the higher immunolabeling with samples prepared without OsO₄, but also the lower membrane preservation under this condition. (C) Relative labeling distribution of NS3 and NS5A. Thawed cryosections of cells post-fixed or not with OsO₄ were labelled with NS3- or NS5A-specific antibodies by using two different blocks and 3 different labeling experiments. Per labeling experiment two grids were considered, counting ∼100–200 gold particles per grid and attributing the particles to the indicated structures. In the case of uninfected cells only background labeling in the cytoplasm, on mitochondria and undefined structures was seen. Numbers refer to the percent of total gold particles counted per sample. ER, endoplasmic reticulum; SV, small vesicles; Cyto, cytosol; Mito, mitochondria; n.d., non-defined structures; NE, nuclear envelope; EE/LE, early/late endosomes; PM, plasma membrane; DMV, double membrane vesicle; LD, lipid droplet.</p