Cytoplasmic localization of core in HCV-infected cells by immunofluorescence without LMB treatment.

<p>(A) <i>Silencing of the proteasome activator PA28γ</i>. Huh 7.5 cells were transfected with an siRNA targeting PA28γ or a control non targeting siRNA 18 h before infection with JFH1. For analysis of the expression of PA28γ by immunofluorescence, cells were stained with rabbit anti-PA28γ antibody, followed by Alexa Fluor 488-conjugated anti-rabbit IgG. Staining for HCV core was carried out 48 h after infection, with the monoclonal anti-core antibody ACAP-27, followed by Alexa Fluor 568-tagged anti-mouse IgG (in red). (a) Non treated HCV (JFH1)-infected Huh 7.5 cells; (b) HCV-infected cells transfected with control, non-targeting siRNA before infection; (c) cells with PA28γ knockdown due to transfection with a specific PA28γ-targeting siRNA. (B) Expression of core in Huh7.5 cells after silencing of the PA28γ proteasome activator. JFH1-infected cells were stained with rabbit anti-lamin B antibody and Alexa Fluor 488-conjugated anti-rabbit IgG as a secondary antibody, to outline the cell nuclei, and with ACAP27 anti-core antibody followed by Alexa Fluor 568-conjugated anti-mouse IgG, for subcellular localization of HCV core. (d) JFH1-infected Huh 7.5 cells without PA28γ silencing, (corresponding to the image shown in (a) panel A); (e) HCV-infected cells transfected with control, non-targeting siRNA before infection (corresponding to the image shown in (b) panel A); and (f) cells with PA28γ knockdown with a PA28γ-specific siRNA before infection with HCV (corresponding to (c) in panel A). Staining of the nuclear membrane with anti-lamin B (green) and with anti-core antibody (red), as described above. No nuclear staining of core was detected, in either siRNA-silenced cells or in cells transfected with a control si-RNA.</p

Identification of a functional, “non-classical” NES” in the core protein.

<p>(A) CLUSTAL W (1.81) software was used for multiple sequence alignment analysis, leading to the identification of a “non classical” NES sequence in domain II of the core protein. The potential NES signal aa(109–133) in core was compared with known viral NES sequences. Underlined regions correspond to hydrophobic amino-acid residues, and letters in bold typeface identify the conserved LXL motifs. The frame delineates a region of the export sequence containing amino-acid residues L(119), I(123) and L(126), which were replaced by alanine residues (the corresponding immunofluorescence analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025854#pone-0025854-g006" target="_blank">Figure 6</a>). (B) The amino-acid sequences of the fragment corresponding to the putative NES aa(109–133) in HCV core proteins are well conserved in different HCV genotypes. The consensus sequences are shown, for each virus genotype, and were obtained by the alignment of 1245 sequences corresponding to the putative NES for HCV type 1A, 2078 sequences for HCV type 1B, and 95, 264, 60, 12 and 121 sequences for HCV types 2, 3, 4, 5 and 6, respectively. Sequences were obtained from the Los Alamos Data Bank (National Institutes of Health). (C) Schematic diagram of the plasmids used to investigate the functionality of the putative export sequence of core, aa(109–133). The SV40 NLS was used as a nuclear reporter, and the NES of the HIV Rev protein was used as a control export signal. The sequences shown were fused to either EGFP or m-Cherry, to allow the visualization of proteins in transfected cells. (D) Subcellular distribution of the proteins encoded by the plasmids depicted above. Huh7 cells grown on coverslips were transfected with the appropriate plasmids; 40 h after transfection, the cells were fixed in 4% PFA and examined by fluorescence microscopy. Panels a-c represent proteins labeled with EGFP, d-f the equivalent proteins labeled with m-Cherry. Proteins containing only the SV40 NLS were present mostly in the cell nuclei (a, d); proteins containing the control SV40 NLS and Rev NES were found in both the nucleus and the cytoplasm (b, e). The core sequence containing a putative NES, aa(109–133), is functional, because it was exported from the nucleus to the cytoplasm (c, f), like HIV Rev NES (b,e). Staining of nuclei with DAPI. (E) Graphical representation of nonparametric one-way ANOVA of the ratios of fluorescence between the nucleus and cytoplasm for the three plasmids. Cytoplasmic fluorescence is significantly higher for both EGFP-NLSSV40-NESRev and EGFP-NLSSV40-core aa(109–133).</p

Nuclear localization of core protein in HCV infection, as demonstrated by electron microscopy.

<p>For electron microscopy analysis, virus particles produced in Huh 7.5 cells were concentrated from the cell supernatant by centrifugation through a sucrose cushion for 4 h at 32,000 rpm in an SW 32 Ti rotor. Concentrated virus preparation was incubated with Huh 7.5 cells at 4°C. The cells were then transferred to 37°C and incubated for a further 20 min. Cells were washed, fixed with 4% PFA and stained with monoclonal anti-core antibody ACAP27, followed by secondary, colloidal gold-labeled anti-mouse IgG. Nanogold staining was enhanced by incubation with the HQ silver enhancement kit, and cells were post-fixed by incubation with 1% osmium tetroxide (for details, see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025854#s2" target="_blank">Materials and Methods</a> section). Non infected cells were also incubated with anti-core antibodies. Panels A, B and C, show representative pictures of HCV core localization in HCVcc-infected cells. The presence of one silver-enhanced gold particle is indicated. LD, lipid droplet; M, mitochondrion; NP nuclear pore. (D) Quantitative evaluation of HCV core labeling (gold particles) in the perinuclear <i>vs</i> other areas of HCV-infected and non infected cells, from randomly selected intracellular zones of equivalent area. Thirty cells were considered for each analysis.</p

Western Blot analyses of constructs used for cell transfection.

<p>The integrity of the constructs used to transfect cells (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025854#pone-0025854-g002" target="_blank">Figure 2</a>) was assessed by Western Blotting. After separation of the proteins in cell lysates by SDS-PAGE, the bands were transferred to nitrocellulose membranes and for detection with anti-EGFP and anti-tubulin antibodies followed by peroxidase-labeled anti-rabbit IgG. The protein bands were detected by chemiluminescence. 1. Lysed Huh 7 cells; 2-Huh 7 cells transfected with pEGFPN1, 3-Huh7 cells transfected with core sequence aa(1-173) inserted into pEGFPN1; 4-Huh 7 cells transfected with the core aa(1–160) sequence in pEGFPN1; Huh 7 cells transfected with core sequence aa(1–140) inserted into pEGFPN1.</p

Nuclear export of core NES is mediated by CRM-1.

<p>Huh7 cells were transfected with a plasmid encoding EGFP-tagged NLS SV40 (a, b), EGFP-tagged NLS SV40 and HIV Rev NES (c, d) or the plasmid encoding EGFP-tagged NLS SV40 and putative core NES, aa(109-133) (e, f). Cells transfected with the EGFP-tagged control plasmid pEGFPC1 are shown (g, h). Twenty-two h after transfection, the cells were treated for 4 h with 10 ng/ml LMB (b, d, f, h; +LMB). The cells were subsequently washed, fixed and analyzed by immunofluorescence microscopy. The bar represents 10 µm.</p

Subcellular localization of HCV core proteins of different lengths in CHO and Huh 7 cells.

<p>Plasmids encoding EGFP-labeled core proteins composed of aa(1–140) (a,d), aa(1–160) (b,c), and aa(1–173) (c,f) were used to investigate the subcellular distributions of the encoded proteins in the several human and non-human cell lines. Cells were transfected with plasmids encoding the corresponding EGFP-labeled proteins, grown for 24 h and analyzed by fluorescence microscopy, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025854#s2" target="_blank">Materials and Methods</a> section. (a–c) results obtained for CHO cell line (the same type of distribution was observed for ARL-6 and HEK-293 cells) and (d–f) distribution representative for Huh 7 cells (and other cells of hepatic origin as HepG2 or Fa2-N4). Bar represents 10 µm.</p

Fluorescence is predominantly nuclear for the protein encoded by the mutated NES.

<p>(A) The subcellular distribution of the proteins encoded by the construct with the mutated nuclear export sequence in HCV core was investigated by immunofluorescence. Mutations were introduced into the NES, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025854#pone-0025854-g005" target="_blank">Figure 5</a> A: amino-acid residues L(119), I(123) and L(126) were replaced by alanine residues (underlined within the frame). (A) Plasmids containing the EGFP, NLSSV40-core aa(109–133) and EGFP NLSSV40-mutated core aa(109–133) sequences were used to transfect Huh 7 cells, as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025854#pone-0025854-g005" target="_blank">Figure 5</a>. After transfection (40 h), the cells were fixed with 4% PFA and examined by fluorescence microscopy. Upper panel represents immunofluorescence analyses of the subcellular distribution of the protein produced by the wild-type EGFP-NLSSV40-core aa(109–133) construct, lower panel shows the distribution of the corresponding construct with a mutated NES. The protein encoded by the wild-type construct (W.T) was found in both the nucleus and the cytoplasm; the protein encoded by the mutated construct (Mut) was found mostly in cell nuclei. Staining of the nuclei with DAPI. (B) EGFP-tagged core protein encoded by the wild type core aa(1–173) construct was found in both the nucleus and the cytoplasm (upper panel); the protein encoded by the mutated construct (Mut) was found mostly in cell nuclei (lower panel). Staining of the nuclei with DAPI. Graphical representation of subcellular distribution of the fluoresecence signal, based on a nonparametric <i>t</i>-test is shown on the right side of each panel. The graph shows the ratios of fluorescence between the nucleus and cytoplasm for the two plasmids encoding the wild-type and mutated proteins.</p

Schematic diagram of structural and functional domains within the HCV core protein.

<p>The RNA-binding region aa(1–57), the three nuclear localization signals (NLS), and the classical NES aa(179–187) and the candidate “non classical” NES aa(109–133) identified in this study are shown. Numbers identify the aa positions covered by each domain and functional region.</p

Subcellular localization of core in LMB-treated cells.

<p>(A) Production of the EGFP-labeled core protein aa(1–173) in Huh7 cells results in the presence of this protein in both the cytoplasmic (a) and nuclear (b) compartments. Huh7 cells were transfected with a plasmid encoding EGFP–labeled aa(1–173) protein and, 40 h later, cells were treated with 10 ng/ml LMB. The inhibition of nuclear export by LMB induced an increase in the accumulation of HCV core protein in the nucleus (c). (B) Subcellular localization of core protein, with and without LMB treatement, confirmed by confocal microscopy analyses. Huh7 cells were transfected with a plasmid encoding the EGFP-labeled core protein aa(1–173), as described in (A). Slides correspond to the panels shown in A: the cytoplasmic (a) and nuclear (b) distribution of the core protein in non treated cells and its nuclear localization after LMB treatment (c). The bar indicates 20 µm.</p