Signal peptide cleavage and internal targeting signals direct the hepatitis C virus p7 protein to distinct intracellular membranes

Abstract

The hepatitis C virus (HCV) p7 protein forms an amantadine-sensitive ion channel required for viral replication in chimpanzees, though its precise role in the life cycle of HCV is unknown. In an attempt to gain some insights into p7 function, we examined the intracellular localization of p7 using epitope tags and an anti-p7 peptide antibody, antibody 1055. Immunofluorescence labeling of p7 at its C terminus revealed an endoplasmic reticulum (ER) localization independent of the presence of its signal peptide, whereas labeling the N terminus gave a mitochondrial-type distribution in brightly labeled cells. Both of these patterns could be visualized within individual cells, suggestive of separate pools of p7 where the N and C termini differed in accessibility to antibody. These patterns were disrupted by preventing signal peptide cleavage. Subcellular fractionation revealed that p7 was enriched in a heavy membrane fraction associated with mitochondria as well as normal ER-derived microsomes. The complex regulation of the intracellular distribution of p7 suggests that p7 plays multiple roles in the HCV life cycle either intracellularly or as a virion component.

FIG. 1.

Characterization of polyclonal rabbit anti-p7 antibody 1055. (A) To test the ability of antibody 1055 (#1055) to specifically detect the p7 protein of HCV, human embryonic kidney 293T cells were transfected with constructs expressing GFP or NS5A-GFP or the HCV proteins E1-E2-p7, fixed, permeabilized, and stained using antibody 1055 (see Materials and Methods). The top row of images show antibody 1055-stained 293T cells expressing GFP with adjacent nontransfected cells that do not give any specific fluorescence for the antibody. The middle two rows of images show 293T cells expressing NS5A-GFP which are also not stained by antibody 1055 but are positive using anti-NS5A (α-5A). The bottom row of images show an example of positive antibody 1055 fluorescence in 293T cells expressing HCV E1-E2-p7 with strong overlap (yellow) between the p7 signal (green) and overlap for E2 stained with ALP98 monoclonal antibody (red). (B) Immunoblots of 293T cell lysates expressing native and tagged variants of p7. BCA-normalized lysates were run on both 10 to 20% MOPS (top) and 15% Tris-glycine (Tris/Gly) gels (bottom two blots) to attempt to resolve size-dependent differences in the electrophoretic mobilities of various p7 variants (see Results) and then immunoblotted using either antibody 1055 or mouse anti-FLAG monoclonal antibody M2 (Sigma). Lanes; 1, mock-transfected cells; 2, p7; 3, FLAG-p7; 4, p7-FLAG; 5, MYC-p7; 6, SP-p7; 7, SPm-p7.

FIG. 2.

p7 stained at the C terminus shows an ER localization independent of the signal peptide. (A) 293T cells were transfected with various p7 expression constructs (see Materials and Methods) and stained using antibody 1055 (#1055) (green) and a marker for the ER, concanavalin A (CON-A) (Texas red [Tx red]). Mock-transfected cells showed no specific staining for p7 (top row), whereas all other p7 expression constructs stained positively with antibody 1055 showing an ER-type localization that significantly overlapped with the concanavalin A signal (shown in yellow in the Merge panels). (B) Mutation of the signal peptidase recognition site for the p7 signal peptide (SPm-p7) gives two phenotypes when stained with antibody 1055; at low levels of expression of SPm-p7, the staining appears as the other p7 constructs in panel A do (top row), whereas more brightly stained cells show cytoplasmic aggregates that appear to contain ER-derived membranes as judged by concanavalin A colocalization (bottom row). (C) The effects of the A743N A745R mutations on signal peptidase activity were assessed using a construct expressing the HCV proteins E1-E2-p7 in which the mutation had been introduced by overlap PCR, pCDNAE1-E2-SPm-p7 (see Materials and Methods). 293T cells transfected with this construct were positive for both antibody 1055 (green) staining of p7 and ALP98 (red) staining of E2, and the staining showed strong colocalization (yellow). Immunoblots of 293T cells expressing E1-E2-p7 via baculovirus transduction (see Materials and Methods) or E1-E2-SPm-p7 using ALP98 (10% Tris-glycine, top gel) and antibody 1055 (12.5% Tris-glycine, bottom gel [dark exposure to show uncleaved E2-p7 in the wild type]) showed the clear abrogation of signal peptidase-mediated cleavage of p7 from E2 in the SPm background.

FIG. 3.

Staining the N terminus of p7 reveals two separate phenotypes in localization. (A) Staining the N terminus of p7-FLAG using an anti-FLAG antibody (α-FLAG) conjugated to FITC reveals two populations of cellular staining phenotypes; more brightly stained cells show a ring-like staining pattern, whereas less brightly stained cells show a more ER-type distribution. The same pattern was observed for cells expressing MYC-p7 stained at the N terminus with 9E10 monoclonal antibody (data not shown). (B) To quantify the apparent difference in staining intensities observed for cells exhibiting the ring-like, mitochondrial p7-FLAG staining pattern (mito) versus the less-intense ER-type distribution (er), 0.2-μm z sections of p7-FLAG-expressing cells stained with FITC-conjugated anti-FLAG antibody were taken at equal exposure and neutral density settings. Individual color channels were defined and subsequently quantified using the Image J program giving a value for mean fluorescence intensity per pixel. The values are the means ± standard errors of the means (error bars) for seven data sets. (C) To confirm that the two patterns of p7-FLAG stained with anti-FLAG antibody (α-FLAG) conjugated to FITC did indeed correspond to true mitochondrial and ER localization, 293T cells were counterstained with markers for the mitochondria and ER, Mitotracker CMXros and concanavalin A (CON-A), respectively (see Materials and Methods). Brightly staining cells (green) showed a clear overlap with the Mitotracker CMXros label (red) and that appeared to concentrate in a ring around the outside of the organelles (top Merge panels), whereas the less bright staining overlapped strongly with the concanavalin A signal (red, bottom Merge panels) and vice versa. Tx red, Texas red.

FIG. 4.

Separate pools of p7 defined by staining the N or C terminus of p7 protein. 293T cells transfected with p7-FLAG were dual labeled using C-terminal antibody 1055 (#1055) detected by an Alexa Fluor 594-conjugated antibody and anti-FLAG antibody (α-FLAG) conjugated to FITC. The same results were obtained again for MYC-p7 stained using anti-MYC monoclonal antibody 9E10 detected by a FITC-conjugated goat anti-mouse secondary antibody (data not shown). (A) p7-FLAG-expressing, bright FITC-labeled cells (green, Merge panel) showed a different staining pattern than the pattern obtained for antibody 1055 (red), and these signals did not significantly overlap. Dim FITC-labeled cells, however, showed colocalization between the signals for the two termini (yellow, Merge panel). (B) To control for artifacts arising from the use of the epitope tags, the same staining protocol was performed on cells expressing pCDNAp7FLAG where the FLAG tag is located at the C terminus. In this case, the signals from both antibodies overlapped independently of the FITC labeling intensity, indicating that the differences observed for p7-FLAG were likely due to differences in the accessibility of the protein termini to the antibody.

FIG. 5.

p7 is enriched in heavy membrane fractions associated with mitochondria. Subcellular fractionation was performed to determine the proportion of p7 in ER- or mitochondrion-associated membrane fractions. (A) 293T cells transfected with various p7 expression constructs were homogenized, and the membranes were separated by differential centrifugation (see Materials and Methods). Pellets obtained at 10,000 × g were designated heavy (H) and contained mitochondria along with associated ER-derived membranes, whereas those obtained at 100,000 × g were designated light (L) ER-derived microsomes. Normalized protein samples from each pellet were then subjected to SDS-PAGE and Western blotting using antibody 1055 (#1055) against p7, the ER marker calreticulin (Cal), or the inner mitochondrial membrane marker cytochrome oxidase subunit 1 (COX). Abbreviations: p7, pCDNAp7; SP-p7, pCDNASP-p7; Fp7, pCDNAFLAG-p7; p7F, pCDNAp7-FLAG; SPmp7, pCDNASPm-p7. (B) Heavy and light membrane fractions were prepared as described above for panel A from 293T cells transduced with baculoviruses expressing the HCV structural proteins. In addition to the markers in panel A, pellets were also Western blotted for the HCV E2 and core proteins (see Materials and Methods). (C) To determine whether p7 associated with mitochondria per se or with membranes associated with these organelles, 293T cells transduced with baculovirus expressing C-E1-E2-p7 were labeled with Mitotracker CMXros prior to homogenization. Crude membrane fractions were generated by differential centrifugation (see Materials and Methods). The 5,000 × g pellet was then pelleted through a sucrose cushion, and mitochondria were purified on a continuous sucrose gradient as judged by Mitotracker CMXros fluorescence (data not shown). Protein content was normalized for all fractions, and samples were subjected to SDS-PAGE and Western blotting as in panel B. Lanes 1, input homogenate; lanes 2, 5,000 × g pellet; lanes 3, 10,000 × g pellet; lanes 4, 100,000 × g pellet; lanes 5 and 6, gradient-purified peak mitochondrial fractions; lanes 7, supernatant after 100,000 × g spin, methanol precipitation, and pelleting (supernatant contains cytosol and residual membranes).

FIG. 6.

Hypothetical model for intracellular targeting of p7. Upon translation, p7 adopts either a single- or double-membrane-spanning topology. A double-membrane-spanning p7 (top left) is more likely to remain in the ER, potentially due to a signal located in the N terminus of the protein. If signal peptide cleavage occurs, double-membrane-spanning p7 will form oligomeric channels, whereas uncleaved E2-p7 is directed into virus particles. A single-membrane-spanning p7 (bottom left) could spontaneously adopt the double-membrane-spanning topology prior to signal peptide cleavage, or by the action of a C-terminal signal, cleaved protein could be targeted to membranes around mitochondria. Upon reaching these membranes, protein would then be free to adopt a double-membrane-spanning topology forming oligomeric channels.