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

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

Subcellular localization of VSV M proteins.

<p>BHK-T7 cells were transfected for 2 h with pTM1 encoding VSV M1 (A-D), M2 (E-F) or M3 (G-H), and fixed 6 h later. Expression and localization of viral proteins were analyzed by immunofluorescence using specific monoclonal antibodies against M1 that also recognize M2 and M3, and the corresponding mouse secondary antibody conjugated to Alexa 488. Localization of M1 in intracellular membranes and dot-like structures at the nuclear envelope or at the cell surface is shown in panels A, B and C, respectively. Localization of M2 and M3 in intracellular compartments (E and G) or surrounding the nucleus (F and H) is shown. Images were acquired with an Axiovert microscope connected to a digital camera.</p

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

Effect of VSV M2 and M3 expression on nucleus-cytoplasm transport of mRNAs.

<p>BHK-T7 cells were transfected for 2 h with pTM1 empty (mock) or pTM1 encoding M1, M2 or M3 proteins. Cells were fixed 6 hpt and <i>in situ</i> hybridization with fluorescein-labeled oligo (dT) probe was carried out to detect cellular mRNAs. VSV M proteins were visualized by immunofluorescence using specific monoclonal antibodies against M1 (αM) and the corresponding mouse secondary antibody conjugated to Alexa 555. To-Pro3 was used as a nuclear marker. Images were acquired with a confocal microscope. Merged images are shown on the right.</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

Induction of vesicle budding from the plasma membrane by VSV M proteins.

<p>BHK-T7 cells were transfected for 2 h with pTM1 encoding M1 (A-F), pTM1 empty (G), M2 (H) or M3 (I). Cells were fixed 6 h after transfection and immunodetection of VSV M proteins was performed using specific monoclonal antibodies and the corresponding mouse-secondary antibodies coupled to gold particles. Cells were visualized with a transmission electron microscope. Arrows indicate sites of vesicle budding at the plasma membrane (A-E) where M1 protein is concentrated, as well as vesicles already released from the cells (F). Statistical analyses of the gold granules distribution was carried out by unpaired (two-tailed) Student <i>t</i>-test. p < 0.01 using the Stata Program Version 11.0.</p

VSV M proteins do not alter cell membrane permeability.

<p>(A) BHK-T7 cells were transfected with pTM1 empty plasmid or pTM1 constructs encoding M1, M2 or M3 proteins. As a positive control, cells were transfected with pTM1-2B (encoding poliovirus 2B viroporin). The medium was removed 2 h later and fresh DMEM containing 5% FCS was added. Cells were pre-treated 15 h after transfection with 0.5 mM of the translation inhibitor hygromycin B (HB) for 15 min. Then, cells were metabolically labelled with [³⁵S]Met/Cys for 45 minutes in the presence or absence of HB. Samples were processed by SDS-PAGE (17.5%), fluorography and autoradiography. (B) BHK-21 cells were mock transfected or electroporated with SV-derived mRNA replicons: Rep C, Rep C+M1 or Rep C+6K, obtained by <i>in vitro</i> transcription from their corresponding DNA templates. Cells were pre-treated with HB and metabolically labeled as indicated in (A). Numbers below each lane indicate the percentages of protein synthesis calculated by dividing the densitometric values for HB-treated cells by the values for untreated cells. A cellular protein band in mock transfected cells, or the band corresponding to the SV C protein in replicon transfected cells, was quantified by densitometric scanning, respectively. Bands corresponding to SV C and VSV M1 protein are indicated with arrows. Detection of α-tubulin served as loading control.</p

Cytotoxic effect mediated by expression of VSV M proteins.

<p>BHK-T7 cells were transfected with pTM1 empty (mock), pTM1-M1, pTM1-M2 or pTM1-M3 for 2 h. Cells were then washed and incubated in DMEM containing 5% FCS until they were fixed at 6, 18 and 24 h post transfection (hpt). Cell morphology was examined with a phase-contrast microscope.</p