NS5A basic cluster mutant is impaired in core envelopment.

<p>(A) Huh7-Lunet cells were transfected with RNA genomes specified in the top: Jc1 wildtype (WT), NS5A R352-355E basic cluster mutant (BCM) and NS5A serine cluster mutant S452/454/457A (SCM). Twelve, 24 and 48 h post transfection cell lysates were prepared and separated by using a 0–30% linear sucrose gradient (left, middle and right panel, respectively). Core protein amounts contained in each fraction were quantified and normalized to total core contained in all fractions. (B) The amounts of E2 (left), NS5A (middle) and ADRP (right) contained in each fraction of cell lysates prepared 48 h post transfection were determined by quantitative Western blot. For each fraction, relative protein amounts are displayed (lower panels). Mean and SEM of two independent experiments are shown. (C) Huh7-Lunet cells were transfected with given HCV genomes and 48 h later cell lysates were prepared and either mock treated or incubated with 15 μg/ml proteinase K (PK) for 40 min on ice. As positive control, samples were treated with 1% Triton X-100 (TX-100) prior to PK digestion. The amount of core protein resistant to PK treatment was determined by Western blot and CMIA (left and right panel, respectively). The graph shows the percentage of PK-resistant core protein. Mean and SEM of three independent experiments is shown. **, <i>p ≤0</i>.<i>01; ***</i>, <i>p ≤0</i>.<i>001</i>.</p

NS5A basic cluster mutant has a defect in core—HCV RNA association.

<p>(A) No impact of a Flag-tag inserted into core protein of a HCV reporter genome (JcR2a) on viral RNA replication and production of infectious virus particles. A schematic representation of the HCV genome indicating the insertion site in the core coding region (codon 2) is given in the top. Huh7-Lunet cells were transfected with the parental renilla luciferase reporter genome (JcR2a) or the variant containing a Flag-tag in core (JcRa2-Flag-core). Left panel: cells were lysed at given time points after transfection and replication was determined by measuring renilla luciferase activity. Right panel: culture supernatants of transfected cells harvested at given time points were used to infect naïve Huh7.5 cells and renilla luciferase activity was measured 72 h later. (B) Core protein amounts contained in transfected cells or released into culture supernatants 72 h after transfection are shown in the left panel (intra- and extracellular, respectively). Amounts of infectious virus released into culture supernatants and specific infectivity of released virus particles are given in the right panel. Mean and SEM of two independent experiments is shown. (C, D) Effect of mutations in NS5A on association of core protein with HCV RNA. Huh7-Lunet cells were transfected with HCV genomes specified in the bottom, 72 h later cells were lysed and Flag-tagged core was enriched by immunoprecipitation (IP) using a Flag-specific monoclonal antibody covalently linked to magnetic beads. (C) Efficiency of IP was determined by core-specific CMIA and normalized to the input. The non-tagged HCV genome (WT) served as technical control for specificity of immunoprecipitation. (D) HCV RNA and GAPDH mRNA (specificity control) coprecipitated with core protein was quantified by RT-qPCR and HCV RNA copy numbers (Y-axis in the left) or relative GAPDH mRNA values (Y-axis in the right) co-precipitated per pg core protein were calculated. Mean and SEM of ten independent experiments is shown. *, <i>p≤0</i>.<i>05; ***</i>, <i>p≤0</i>.<i>001</i>.</p

Hypothetical roles of serine- and basic clusters in NS5A domain III for the assembly of infectious HCV particles.

<p>(A) NS5A interacts with core protein to recruit (RNA-containing) replication complexes (RCs) to HCV assembly sites where Core, E1, E2, p7 and NS2 reside. NS5A delivers the viral genome, eventually in conjunction with NS3 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005376#ppat.1005376.ref078" target="_blank">78</a>], from the RCs to core protein to trigger genome encapsidation and nucleocapsid formation. This induces their budding into the ER lumen that might be promoted by the envelope glycoproteins E1 and E2 in conjunction with p7 and NS2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005376#ppat.1005376.ref011" target="_blank">11</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005376#ppat.1005376.ref019" target="_blank">19</a>]. (B) The NS5A basic cluster (BC) mutant is able to recruit the RCs to the assembly site by interacting with core protein, but is unable to associate with viral RNA for the assembly process. (C) The NS5A serine cluster (SC) mutant does not affect NS5A-HCV RNA association, but fails to interact with core protein and therefore the RCs are not recruited to the assembly site. As a result NS5A and core protein accumulate around distinct cytosolic lipid droplets (cLDs). Overall, both NS5A mutants are unable to load core protein with viral RNA, although for different reasons, and as a consequence envelopment of nucleocapsids is impaired.</p

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

<div><p>Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.</p></div

Mutations affecting the NS5A basic cluster reduce interaction with HCV RNA.

<p>(A) Schematic representation of the Jc1 genome containing a Flag-tag in NS5A DII. Huh7-Lunet cells were transfected with HCV genomes specified in the bottom and 72 h later cells were lysed and NS5A was enriched by immunoprecipitation (IP) using a Flag-specific monoclonal antibody covalently linked to magnetic beads. Captured NS5A proteins were separated by electrophoresis into an 8% acrylamide gel and analyzed by Western blot using a mono-specific NS5A antibody. The same amounts of input proteins were loaded onto the gel in parallel. The efficiency of the Flag-NS5A IP was determined by quantifying the bands and normalizing to the input signals. The non-tagged HCV genome (WT) served as technical control for specificity of immunoprecipitation. (B) Quantification of HCV RNA co-precipitated with Flag-NS5A. HCV RNA and GAPDH mRNA (specificity control) was determined by RT-qPCR. The percentage of viral RNA copies co-precipitated (co-IP) with NS5A was determined and used to calculate the enrichment of HCV RNA co-precipitated with NS5A. Note that GAPDH mRNA was below the detection limit in the co-precipitated samples and therefore is not displayed. Mean and SEM of six independent experiments is shown. *, <i>p ≤0</i>.<i>05; **</i>, <i>p ≤0</i>.<i>01; not significant (NS)</i>, <i>p ≥0</i>.<i>05</i>.</p

Impact of mutations affecting the basic cluster motif in NS5A DIII on RNA replication and virus production.

<p>(A) Schematic representation of NS5A domains: amphipathic α-helix (AH); domain (D) I, II and III; low complexity sequences (LCS) 1 and 2. The positions of the hemagglutinin (HA) epitope tag inserted within DII and the serine cluster (SC; S452/454/457) in DIII are indicated on the top. An alignment of the amino acid sequence of the NS5A basic cluster motif of several HCV isolates belonging to genotype 1 to 7 is given at the bottom. Numbers refer to amino acid residues 352 to 355 of the JFH1 isolate (corresponding to polyprotein residues 2328 to 2331). *, invariant amino acid residue across the displayed HCV isolates;:, conservation of physicochemical properties of the amino acid. The following HCV genomes were used for the alignment (gene bank accession numbers are given in parenthesis): H77 (AF009606), Con1 (AJ238799), Ad78 (AJ132997), J6 2a (Af177036), 452 (DQ437509), ED43 (Y11604), SA13 (AF064490), 6a33 (AY859526), QC69 (EF108306) and JFH1. (B) Given glutamic acid residue substitutions were inserted into a subgenomic JFH1 Firefly luciferase reporter replicon (sgJFH1-Fluc; top panel) and replication kinetics were determined. The various sgJFH1 constructs were transfected into Huh7-Lunet cells and harvested 4, 12, 18, 24, 48 and 72 h later. Luciferase activity was quantified and values were normalized to the respective 4 h-value. Mean and SEM of three independent experiments are shown. Background was determined with an RdRp-defective mutant (GDD). (C) The same mutations were introduced into the full-length HCV chimera Jc1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005376#ppat.1005376.ref004" target="_blank">4</a>] shown in the top and 24, 48 and 72 h after transfection into Huh7-Lunet cells virus amounts contained in culture supernatants were quantified by limiting dilution assay. Values were normalized to the wildtype (WT) virus that was set to 100%. Mean and SEM of three independent experiments are shown. Background of the assay was determined with a deletion mutant lacking the envelope glycoprotein coding region (ΔE1E2). (D) NS5A amounts contained in cells 72 h after transfection with the Jc1 variants were determined by Western Blot with NS5A-specific antibodies; ß-actin served as loading control. Numbers in the left refer to apparent molecular weights of marker proteins in kilo Dalton (KDa).</p

Mutations disrupting the NS5A basic cluster increase E2 accumulation in doughnut-like structures without affecting NS5A –core protein colocalization.

<p>Huh7-Lunet cells were transfected with the HCV genomes specified in the left and fixed 48 h later. HCV proteins were detected by immunofluorescence using mono-specific antibodies. (A) Representative images showing E2—core protein subcellular distribution. Images were generated with a spinning disk confocal microscope and deconvolved to generate a 3D reconstruction with deep projection (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005376#sec010" target="_blank">Materials and Methods</a>). The location of the cropped sections is indicated with white boxes in each overview panel. Scale bars represent 10 μm for overviews and 1 μm for cropped sections. (B) Quantification of core and E2 structures per cell. (C) Amounts of E2, NS5A and core protein were determined by Western blot using mono-specific antibodies; ß-actin served as loading control. (D) Representative images showing core—NS5A co-localization and their proximity to LDs (stained with BODIPY). Scale bars represent 10 μm for overviews and 5 μm for cropped sections. (E) Degree of NS5A - core co-localization as determined by Pearson correlation coefficient. Each dot represents one cell. For each HCV construct, the mean and SD of n = 100 is shown.</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

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 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