Journal:

Article Title: Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

doi: 10.1128/JVI.76.8.3720-3730.2002

Figure Lengend Snippet: p7 is an integral membrane protein. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinant at a multiplicity of infection of 5 PFU/cell. At 5 h postinfection, membrane fractions were prepared as described in Materials and Methods and treated with 0.1 M sodium carbonate, pH 11.3. After separation of membrane-bound (M) and soluble (S) proteins, the presence of p7NT or p7CT in these fractions was revealed by Western blotting with the anti-Myc MAb. Sizes (in kilodaltons) of protein molecular mass markers are indicated on the left.

Article Snippet: Monoclonal antibodies (MAbs) A4 (anti-E1 [ 12 ]), OKT4 (anti-CD4; ATCC CRL-8002 [ 42 ]), and MYC 9E10 (anti-Myc; ATCC CRL-1729 [ 15 ]) were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer.

Techniques: Membrane, Virus, Recombinant, Infection, Western Blot

Journal:

Article Title: Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

doi: 10.1128/JVI.76.8.3720-3730.2002

Figure Lengend Snippet: CD4-p7 protein is exported to the cell surface. HepG2 cells were coinfected with vTF7-3 and a vaccinia virus recombinant expressing the ectodomain of CD4 in fusion with p7 (A) or a truncated form of HCV polyprotein (CE1E2p7CT) (B) at a multiplicity of infection of 3 PFU/cell. Cells were fixed with paraformaldehyde at 6 h postinfection, permeabilized or not with Triton X-100, immunostained with the anti-CD4 MAb OKT4 (A) or anti-Myc antibody (B), and analyzed by immunofluorescence.

Article Snippet: Monoclonal antibodies (MAbs) A4 (anti-E1 [ 12 ]), OKT4 (anti-CD4; ATCC CRL-8002 [ 42 ]), and MYC 9E10 (anti-Myc; ATCC CRL-1729 [ 15 ]) were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer.

Techniques: Virus, Recombinant, Expressing, Infection, Immunofluorescence

Journal:

Article Title: Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

doi: 10.1128/JVI.76.8.3720-3730.2002

Figure Lengend Snippet: Analysis of the endo H sensitivity of CD4-p7. HepG2 cells were coinfected with vTF7-3 and a vaccinia virus recombinant expressing CD4 or CD4-p7 at a multiplicity of infection of 5 PFU/cell. At 4.5 h postinfection, infected cells were pulse-labeled for 10 min and chased for the indicated times (in hours). Cell lysates were immunoprecipitated with the anti-CD4 MAb and treated or not with endo H. Proteins were separated by SDS-PAGE (10% polyacrylamide). Endo H-resistant proteins are indicated by asterisks. The size (in kilodaltons) of a protein molecular mass marker is indicated on the left.

Article Snippet: Monoclonal antibodies (MAbs) A4 (anti-E1 [ 12 ]), OKT4 (anti-CD4; ATCC CRL-8002 [ 42 ]), and MYC 9E10 (anti-Myc; ATCC CRL-1729 [ 15 ]) were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer.

Techniques: Virus, Recombinant, Expressing, Infection, Labeling, Immunoprecipitation, SDS Page, Marker

Journal:

Article Title: Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

doi: 10.1128/JVI.76.8.3720-3730.2002

Figure Lengend Snippet: Identification of a signal sequence in the C-terminal half of p7. (A) Sequence analysis of the C-terminal transmembrane domain of the p7 used in this work (HCV H strain), indicating that this domain has the characteristic structural features of a signal peptide (54). A typical signal peptide is composed of an N-terminal region (n-domain) encompassing between one and three positively charged residues (K33 and R35 here), a hydrophobic core region (h-domain) forming an α-helix (segment 41 to 57), and a more polar, flexible region (c-domain) containing the signal peptidase cleavage site (segment 58 to 63). Residues at positions −1 and −3 relative to the cleavage site are small neutral residues (A63 and A61) and form the recognition site for signal peptidase (53). Furthermore, an α-helix-destabilizing residue is frequently located at position −6 (P58) and/or in the middle of the h-domain (P49). The black box indicates the predicted minimal transmembrane segment (see Fig. ​Fig.1E).1E). (B) Schematic representation of the proteins used to identify the signal sequence function of the C-terminal half of p7. Sp1E1 corresponds to E1 with its signal sequence, and SpNS2E1 corresponds to the C-terminal half of p7 (residues 781 to 809 on the polyprotein) followed by the ectodomain and transmembrane domain of E1. (C) The C-terminal transmembrane half of p7 is a signal sequence. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinant at a multiplicity of infection of 5 PFU/cell. At 4.5 h postinfection, cells were labeled for 1 h with 35S-Protein Labeling Mix. Cell lysates were immunoprecipitated with MAb A4 (anti-E1) and treated or not with endo H. Samples were analyzed by SDS-PAGE (12% polyacrylamide) and autoradiography. Sizes (in kilodaltons) of protein molecular mass markers are indicated.

Article Snippet: Monoclonal antibodies (MAbs) A4 (anti-E1 [ 12 ]), OKT4 (anti-CD4; ATCC CRL-8002 [ 42 ]), and MYC 9E10 (anti-Myc; ATCC CRL-1729 [ 15 ]) were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer.

Techniques: Sequencing, Residue, Virus, Recombinant, Infection, Labeling, Immunoprecipitation, SDS Page, Autoradiography

Journal:

Article Title: Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

doi: 10.1128/JVI.76.8.3720-3730.2002

Figure Lengend Snippet: Determination of the topology of p7 by epitope tagging. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinant at a multiplicity of infection of 3 PFU/cell and analyzed by indirect immunofluorescence. Cells were fixed with paraformaldehyde at 6 h postinfection, permeabilized or not with Triton X-100 (A) or digitonin (B), and immunostained with the anti-Myc MAb. A schematic representation of the topology of the different p7 polypeptides tagged with a Myc epitope is presented on the left.

Article Snippet: Monoclonal antibodies (MAbs) A4 (anti-E1 [ 12 ]), OKT4 (anti-CD4; ATCC CRL-8002 [ 42 ]), and MYC 9E10 (anti-Myc; ATCC CRL-1729 [ 15 ]) were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer.

Techniques: Virus, Recombinant, Infection, Immunofluorescence