Ultrastructural Localization and Molecular Associations of HCV Capsid Protein in Jurkat T Cells

Figure 1. Transmission Electron Microscopy of HCV core-GFP (A–F) or GFP (G,H) transduced Jurkat cells. Images (A) (x20000), (B) (x12000), and (C) (x7000) are representative micrographs from GFP HCV core - expressing Jurkat cells. Gold particles indicate the position of immunolabeled HCV core-GFP inside Jurkat cells. Immunostaining (arrows) revealed GFP HCV core protein in the nucleus [N] (B), cytoplasm [C] (A) and mainly in the nucleoli [Nl] (B,C). Micrographs (D) (x20000), (E) (x12000), and (F) (x30000) show a magnification of the gold labeled HCV core-GFP expressing Jurkat cell. This closer inspection confirmed gold particles labeling GFP core protein (arrows) in the nucleus, with enrichment and a clustering distribution in the nucleoli. Images (G) (x20000) and (H) (x20000) are representative micrographs of GFP-expressing control Jurkat cells. Gold particles indicate the position of immunolabeled GFP. Immunostaining (arrows) revealed GFP in the cytoplasm (G) and nucleus (G,H). No immunostaining was observed in the nucleoli in GFP control cells (G,H).

Figure 2. Subcellular localization of proteins associated with HCV core protein. Associated proteins were classified by String™ 10 software using the “actions” view, according to the “GO cellular components” distribution option, in order to identify their subcellular localization. String analysis reported that proteins were nuclear (A) (128 from 222), nucleolar (B) (43 from 222), and cytoplasmic (C) (150 from 222). Colored lines between proteins indicate the type of evidence for each interaction, with a minimum required confidence score of 0.4.

Figure 3. Schematic representation of the identified proteins involved in different top networks categories. The identified proteins were associated with splicing and processing of RNA (A); cell cycle progression, proliferation and apoptosis (B); infection by RNA viruses, replication of RNA viruses and Hepatitis C virus infection (C). Protein shapes indicate their function. Proteins are localized in the nucleus (orange), nucleolus (violet), cytoplasm (blue), and plasma membrane (yellow), according to String 10 Go Cellular component. All the relationship lines are supported by at least one reference from the literature, textbooks or canonical information stored in the Ingenuity Knowledge Base.

Figure 4. HCV Core protein interacts with PP1γ and MYPT1 in PBMC blasts. Postnuclear lysates from Human PBMCs stimulated with PHA and cultured during 5 days with IL-2, were subjected to HCV Core protein pull down. Proteins pulled down by HCV Core coated magnetic beads (PBMC Core band) or by uncoated magnetic beads (PBMC beads band) were analyzed by Western blot with anti-human MYPT1 (A) and anti-human PP1γ (B). Molecular mass, in kDa, is indicated on the side and band quantitation at the bottom for each Western blot.

Figure 5. Predicted Nucleolar localization sequences in HCV core protein. Graph obtained from the Nucleolar Localization Sequence detector web server (NoD), displaying NoLS prediction score for each residue of HCV Core protein. Pink shaded regions represent the range of scores within which a 20-residues segment is predicted to be a NoLS. Thus, pink shaded regions represent the NoLS candidate segment, which highlights scores above 8.0.