signal peptide cleavage and internal targeting signals direct the

JOURNAL OF VIROLOGY, Dec. 2005, p. 15525–15536 Vol. 79, No. 240022-538X/05/$08.00�0 doi:10.1128/JVI.79.24.15525–15536.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Signal Peptide Cleavage and Internal Targeting Signals Direct theHepatitis C Virus p7 Protein to Distinct Intracellular MembranesStephen Griffin, Dean Clarke, Christopher McCormick,† David Rowlands, and Mark Harris*

Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology,University of Leeds, Leeds LS2 9JT, United Kingdom

Received 8 February 2005/Accepted 30 August 2005

The hepatitis C virus (HCV) p7 protein forms an amantadine-sensitive ion channel required for viralreplication in chimpanzees, though its precise role in the life cycle of HCV is unknown. In an attempt to gainsome insights into p7 function, we examined the intracellular localization of p7 using epitope tags and ananti-p7 peptide antibody, antibody 1055. Immunofluorescence labeling of p7 at its C terminus revealed anendoplasmic reticulum (ER) localization independent of the presence of its signal peptide, whereas labeling theN terminus gave a mitochondrial-type distribution in brightly labeled cells. Both of these patterns could bevisualized within individual cells, suggestive of separate pools of p7 where the N and C termini differed inaccessibility to antibody. These patterns were disrupted by preventing signal peptide cleavage. Subcellularfractionation revealed that p7 was enriched in a heavy membrane fraction associated with mitochondria as wellas normal ER-derived microsomes. The complex regulation of the intracellular distribution of p7 suggests thatp7 plays multiple roles in the HCV life cycle either intracellularly or as a virion component.

Hepatitis C virus (HCV) is a major cause of chronic liverdisease worldwide and is now the most common reason forliver transplants in Western countries. Unusually for an RNAvirus, the majority of patients become persistently infectedafter a mild acute episode, and clinical intervention occurs inlate-stage symptomatic patients. Current therapy compriseshigh-dose pegylated alpha interferon (IFN) combined with theguanosine analogue ribavirin. The efficacy of this regimen islargely dependent on the viral genotype; the most prevalentgenotype 1 viruses possess high levels of innate resistance toIFN, and reservoirs of resistance in other genotypes are build-ing due to the highly variable nature of HCV (40). Interest-ingly, a recent meta-analysis of clinical trials in which patientswere treated with a triple combination of IFN, ribavirin, andamantadine showed that this approach gave improved sus-tained viral responses in patients that previously did not re-spond to dual therapy, most often those infected with genotype1 HCV (16).

HCV has a single-stranded positive-sense RNA of around9.6 kb and is the prototype member of the Hepacivirus genus ofthe Flaviviridae family (13, 35). While, until very recently, norobust in vitro replication system has existed for HCV (27, 43,46), many functions of the viral nonstructural proteins havebeen elucidated using replicons (7, 8, 23, 28, 33). The inabilityof these systems to produce extracellular virus has limitedstudies on structural proteins to virus-like particles (VLPs)made either within insect cells (4, 15) or by mammalian ex-pression (5, 6), or more recently, to the use of pseudotypedretrovirus systems to investigate receptor tropism and cell en-try (2, 3, 22).

The p7 protein of HCV is not required for RNA replicationor the formation of VLPs in insect cells, and it is uncertainwhether it is a virion component. p7 is a small hydrophobicprotein of 63 amino acids located within the HCV genome atthe junction between the structural and nonstructural proteins(26, 30). We previously showed that p7 from genotype 1b HCVforms an oligomeric ion channel in planar lipid bilayers thatcan be blocked by amantadine at micromolar concentrations,leading to our proposal that the potential antiviral effect ofamantadine described above may be due to its action on p7(19). Others have subsequently confirmed this ion channelactivity for different HCV genotypes and have identified otherchannel-blocking compounds (32, 34). The important findingthat p7 is required for replication of HCV in chimpanzeesconfirms the protein as a target for antiviral chemotherapy, yetits role in viral replication is unknown (38). The homologousp7 protein from bovine viral diarrhea virus is known to benecessary for the generation of infectious virus particles,though whether virions are able to assemble or are secreted inan immature form is not known, and attempts to detect theprotein in purified particles were unsuccessful (18, 21). Fur-thermore, preventing the already inefficient cleavage of p7from its precursor E2-p7 had a similar deleterious effect onvirus spread, though whether this occurred at the same point inthe virus life cycle is unknown. A role in assembly for HCV p7is also suggested by our finding that p7 was able to replace theinfluenza A virus M2 protein in maintaining the pH-sensitive,receptor-binding conformation of the viral hemagglutinin dur-ing transport to the cell surface (20). The apparent localizationof p7 to mitochondrial membranes in our study, however,seemed counterintuitive given its ability to replace M2 and wascontrary to the findings of other investigators that p7 localizedto the endoplasmic reticulum (ER) of transfected cells (11).This paradox is important to resolve, as localization could givevital clues to the function of p7.

Here, we have combined indirect immunofluorescence with

* Corresponding author. Mailing address: Institute of Molecularand Cellular Biology, Faculty of Biological Sciences, University ofLeeds, Leeds LS2 9JT, United Kingdom. Phone: 44 (113) 343-5632.Fax: 44 (113) 343-5638. E-mail: [email protected].

† Present address: Molecular Biology, University of Southampton,Southampton SO16 6 YD, United Kingdom.

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subcellular fractionation to clarify the intracellular localizationof HCV p7. We have examined the effect of the upstreamsignal peptide from E2 on the distribution of p7 using a novelrabbit polyclonal antibody to the C terminus of the protein incombination with epitope-tagged proteins. We show that na-tive or tagged p7 is able to target the ER independently of itssignal peptide, presumably in a posttranslational manner. Forcells expressing native or epitope-tagged p7, we have identifieda population of brightly labeled cells that contain two separatepools of p7 differing in the accessibility of their N or C terminito antibody: N-terminally stained p7 localizing in ER mem-branes associated with mitochondria and C-terminally stainedmolecules residing in the normal ER. Fractionation of cellularhomogenates confirms that p7 localizes both to the normal ERand also to a heavy membrane fraction associated with mito-chondria.

MATERIALS AND METHODS

Plasmid constructs. pCDNAp7 and pCDNAFLAGp7 have been described pre-viously (20). PCR amplimers for pCDNASP-p7, pCDNASPm-p7, pCDNAMYCp7,and pCDNAp7FLAG were generated using Vent DNA polymerase (New En-gland BioLabs, Inc.) with the J4 infectious molecular clone pCVJ46LS as thetemplate (45). Specific primers generated amplimers corresponding to positions2526 to 2768 for SP constructs (containing signal peptide) or positions 2580 to2768 for p7 open reading frame constructs, and epitope tags were incorporatedat either terminus by modifying the appropriate primer. All amplimers forpCDNA constructs were digested with EcoRI and NotI and ligated into anappropriately digested vector, pCDNA3.1 (Invitrogen). The SPm construct mu-tations A743N A745R were generated using a long forward primer containingthe mutation in its sequence. Primers containing these mutations were also usedto generate pCDNAE1-SPmp7 by overlap PCR of two amplimers: the first fromthe E1 signal peptide (the same as that used to generate the E1-p7 baculovirus[see below]) to the mutated N terminus of p7 and the second from the mutatedN terminus of p7 to the same p7 reverse primer used above. All constructs wereverified by double-stranded DNA sequencing. Primer sequences are available onrequest.

Recombinant baculoviruses. Baculoviruses expressing the HCV structuralproteins were generated using the Bac-2-Bac baculovirus expression system (In-vitrogen) incorporating a modified transfer vector containing a constitutive mam-malian promoter sequence (CAG promoter comprising the cytomegalovirusimmediate-early enhancer controlling the chicken �-actin promoter from pBac-Mam-2 [Novagen]), pFBM. HCV sequences from core to p7 or E1 to p7 (incor-porating the E1 signal peptide) were amplified using specific primers (sequencesavailable on request) and cloned into pFBM digested with EcoRI. Viruses wereamplified in and virus titers were determined on Spodoptera frugiperda Sf-9 cellsand used at 107 PFU/ml for transduction of 293T cells seeded the previous dayat 1 � 106 cells in a 10-cm dish.

Mammalian cell culture and transfection. Human embryonic kidney 293Tcells and African green monkey kidney Vero cells were passaged in Dulbecco’smodified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum,100 IU/ml penicillin, and 100 �g/ml streptomycin. Human hepatoblastomaHepG2 cells were passaged in minimal essential medium (Invitrogen) supple-mented with 10% fetal calf serum, 100 IU/ml penicillin, 100 �g/ml streptomycin,5 mM glutamine, and nonessential amino acids.

All transfections were carried out using Lipofectamine (Invitrogen) at 5 �l/�gDNA according to the manufacturer’s instructions, overnight in Optimem (In-vitrogen) serum-free medium. Cells for fluorescence analysis were mounted onpoly-L-lysine-coated glass coverslips in 12-well plates at approximately 4 � 104

cells/coverslip and transfected with 0.5 �g DNA. Cells to be transfected forheavy/light membrane separation were seeded at approximately 2 � 105 cells/well of a six-well plate and transfected as described above with 1 �g DNA.

Antibodies. Antibody 1055 affinity-purified rabbit polyclonal antibody wasraised against a peptide representing the C-terminal six residues of p7 from theJ4 infectious clone of HCV genotype 1b, PPRAYA. The peptide CGGGPPRAYA was coupled to tuberculin purified protein derivative, and four rabbitswere immunized and then boosted four times. Rabbit 1055 gave positive resultsin an enzyme-linked immunosorbent assay against the immunogen peptide andalso recombinant histidine-tagged glutathione S-transferase–p7 prepared as de-scribed previously (19) using preimmune serum as an animal-specific negative

control. The final working antibody was affinity purified with peptide to a con-centration of 307 �g/ml. Antibody 1055 was detected by indirect fluorescenceusing Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor 594-conjugatedchicken anti-rabbit secondary antibodies (Molecular Probes). FLAG-tagged pro-teins were detected by indirect fluorescence using a fluorescein isothiocyanate(FITC)-conjugated mouse anti-FLAG monoclonal antibody, M2 from Sigma.MYC-tagged protein was detected using mouse monoclonal antibody 9E10 (12)and a FITC-conjugated goat anti-mouse secondary antibody (Sigma). Mousemonoclonal antibody to HCV E2 protein, ALP98 was a gift from Arvind Patel(MRC Virology Unit, Glasgow, United Kingdom) and was detected by fluores-cence using an Alexa Fluor 594-conjugated goat anti-mouse secondary antibody.The rabbit anti-NS5a antiserum was a gift from Ralf Bartenschlager and wasdetected using the same Alexa Fluor 594-conjugated chicken anti-rabbit second-ary antibody described above. HCV core protein was detected using a mousemonoclonal antibody 215/07 from Biogenesis. A mouse monoclonal antibody tocytochrome oxidase (Oxphos IV) subunit 1 was obtained from Molecular Probes,and a rabbit polyclonal antiserum to human calreticulin was from Calbiochem.Detection of antibodies on Western blots was achieved using appropriate horse-radish peroxidase-conjugated secondary antibodies (Sigma).

Indirect fluorescence detection of HCV p7. Cells to be labeled with Mito-tracker CMXros (Molecular Probes) were incubated in a 200 nM solution of thedye in Dulbecco’s modified Eagle medium for 1 h prior to fixation at 16 hposttransfection. Cells transfected on coverslips were washed three times inphosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 20min at room temperature, and then washed twice more in PBS. Cells to beanalyzed by immunocytochemistry were permeabilized with 0.1% Triton X-100in PBS for 5 min. Coverslips were washed three times and then incubated withprimary antibody diluted in PBS containing 10% fetal calf serum for 1 h at roomtemperature in a dark humidified container. Coverslips were washed three moretimes and incubated with the appropriate secondary antibody under the sameconditions along with, where appropriate, Texas Red-conjugated concanavalin A(Molecular Probes) as a marker for the ER. Nuclei were labeled with Hoechststain or TRO-PRO 3 (Molecular Probes) diluted 1/10,000 in PBS. The cells werethen washed three times in PBS and once in distilled water prior to analysis.Images were captured using a DeltaVision restoration system (Applied Preci-sion, Inc., Issaquah, WA) and an Olympus IX-70 inverted microscope. Opticalsections of 0.2 microns were captured with a CoolSNAPHQ charge-coupled de-vice camera (Roper Scientific, Tucson, AZ). Digital deconvolution and imageanalyses were then performed on three-dimensional data sets using 15 iterationsof a constrained iterative deconvolution algorithm with SoftWoRx deconvolutionsoftware (Applied Precision, Inc.). Subsequent quantification of FLAG fluores-cence from images of equal exposure times and neutral density filter settings wasperformed on individual color channels from deconvoluted sections using theImage J program; an average of seven individual images was taken.

Subcellular fractionation. 293T cells were washed three times in PBS, thenscraped into 1 ml of ice-cold fractionation buffer (5 mM HEPES [pH 6.8], 1 mMEDTA, 250 mM sucrose, protease inhibitors), and homogenized on ice with 50strokes of a loose-fitting Dounce homogenizer. Homogenates were then clearedof nuclei and unbroken cells by spinning at 1,000 � g in a microcentrifuge at 4°Cfor 5 min. For separation of crude heavy mitochondrial and light microsomalmembranes, homogenates were spun in a microcentrifuge at 10,000 � g for 10min at 4°C to obtain the heavy membrane pellet. This pellet was then washedtwice by resuspension in 0.5 ml fractionation buffer and respinning. The super-natant from the first spin was respun at 10,000 � g for another 10 min to removeany residual mitochondria. This clarified supernatant was then spun in a SorvallS100 AT3 rotor at 100,000 � g for 1 h to obtain a microsomal pellet. Both pelletswere lysed in 50 �l of EBC lysis buffer (50 mM Tris-HCl [pH 8.0], 140 mM NaCl,100 mM NaF, 200 �M Na3VO4, 0.1% sodium dodecyl sulfate [SDS], 0.5%NP-40), and protein concentrations were normalized by the bicinchoninic acid(BCA) protein assay (Bio-Rad) prior to SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting.

To prepare purified mitochondria, cleared homogenates from cells previouslylabeled with Mitotracker CMXros (see above) were pelleted first at 5,000 � g,then at 10,000 � g, and finally at 100,000 � g and washed as described above.Pellets were resuspended in fractionation buffer such that input samples for eachstage could be taken (550 �l for the 5,000 � g pellet; other pellets in 100 �l). Fivehundred microliters of the 5,000 � g pellet suspension was then layered over a20 to 50% weight/volume continuous sucrose gradient cushion in 5 mM HEPES(pH 6.8) and spun at 52,000 � g in a Sorvall AH650 rotor for 45 min to pelletmitochondria and remove attached membranes. The pellet was then resus-pended in fractionation buffer and layered over a 20 to 50% weight/weightcontinuous sucrose gradient in 5 mM HEPES (pH 6.8) and spun under the sameconditions. The gradient was harvested using a cannula into 12 equal fractions.

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Fifty microliters of each sample was then analyzed by fluorimetry using a stan-dard rhodamine filter set to detect the peak fluorescence of MitotrackerCMXros. Peak samples were then diluted to a sucrose concentration of 250 mMand spun at 20,000 � g for 30 min to pellet mitochondria in a microcentrifuge at4°C. This pellet was then washed in 0.5 ml fractionation buffer and respun at6,500 � g at 4°C for 15 min to give the final mitochondrial pellet. Pellets from allstages of purification were resuspended in 60 �l fractionation buffer, and proteinconcentrations were normalized with input samples by BCA prior to SDS-PAGEand Western blotting.

RESULTS

Characterization of a novel anti-p7 peptide antiserum, an-tibody 1055. We previously generated small quantities of amouse antipeptide antiserum to the N terminus of p7 that wehave since exhausted (19). We therefore attempted to generatenew antibodies against either terminus of the protein and weresuccessful in producing a rabbit polyclonal antibody, antibody1055, against a peptide corresponding to the C-terminal sixamino acids (PPRAYA) of p7 from the J4 infectious clone ofHCV genotype 1b (45). To characterize this antibody for use indetecting p7 by immunofluorescence, human embryonic kid-ney 293T cells expressing green fluorescent protein (GFP), anHCV NS5A-GFP fusion protein, or p7 (in the context of E1-p7in the example shown) were fixed, permeabilized, and stainedwith antibody 1055 as described in Materials and Methods(Fig. 1A). Antibody 1055 showed no cross-reactivity with cel-lular or expressed protein in all cell types tested except whenp7 was present. When expressed with HCV E1 and E2, the p7signal overlapped significantly with the staining for E2 andshowed localization consistent with ER membrane association.p7 was also specifically detected when expressed alone in var-ious forms (see below and see Fig. 2). In addition to 293T cells,p7 was readily detectable when HepG2 or Vero cells were used(data not shown), though the level of expression and efficiencyof transfection were significantly reduced in these cells, requir-ing coinfection with recombinant fowlpox virus expressing T7polymerase. For this reason, the results hereafter compriseimages and blots from 293T cells, though the other cell linesshowed similar phenotypes in all cases.

We also used antibody 1055 to detect p7 expression by im-munoblotting (Fig. 1B). p7 was expressed in 293T cells bothwith and without its upstream signal peptide, with epitope tagsat either terminus, or where the signal peptidase recognitionsite had been mutated (see Materials and Methods). Interest-ingly, the migration of p7 on gels did not appear to directlycorrespond with the expected size of the protein, and this wasobserved for both 10 to 20% morpholinepropanesulfonic acid(MOPS) gradient gels and 15% Tris-glycine gels. Detectionwith antibody 1055 showed that the presence of an N-terminalepitope tag in place of the signal peptide (Fig. 1B, lanes 3 and

FIG. 1. Characterization of polyclonal rabbit anti-p7 antibody1055. (A) To test the ability of antibody 1055 (#1055) to specificallydetect the p7 protein of HCV, human embryonic kidney 293T cellswere transfected with constructs expressing GFP or NS5A-GFP or theHCV proteins E1-E2-p7, fixed, permeabilized, and stained using anti-body 1055 (see Materials and Methods). The top row of images showantibody 1055-stained 293T cells expressing GFP with adjacent non-transfected cells that do not give any specific fluorescence for theantibody. The middle two rows of images show 293T cells expressingNS5A-GFP which are also not stained by antibody 1055 but are pos-itive using anti-NS5A (�-5A). The bottom row of images show anexample of positive antibody 1055 fluorescence in 293T cells express-ing 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 andtagged variants of p7. BCA-normalized lysates were run on both 10 to20% MOPS (top) and 15% Tris-glycine (Tris/Gly) gels (bottom twoblots) to attempt to resolve size-dependent differences in the electro-phoretic mobilities of various p7 variants (see Results) and then im-munoblotted using either antibody 1055 or mouse anti-FLAG mono-clonal 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.

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FIG. 2. p7 stained at the C terminus shows an ER localization independent of the signal peptide. (A) 293T cells were transfected with variousp7 expression constructs (see Materials and Methods) and stained using antibody 1055 (#1055) (green) and a marker for the ER, concanavalinA (CON-A) (Texas red [Tx red]). Mock-transfected cells showed no specific staining for p7 (top row), whereas all other p7 expression constructsstained positively with antibody 1055 showing an ER-type localization that significantly overlapped with the concanavalin A signal (shown in yellowin the Merge panels). (B) Mutation of the signal peptidase recognition site for the p7 signal peptide (SPm-p7) gives two phenotypes when stainedwith 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 morebrightly 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 HCVproteins E1-E2-p7 in which the mutation had been introduced by overlap PCR, pCDNAE1-E2-SPm-p7 (see Materials and Methods). 293T cellstransfected with this construct were positive for both antibody 1055 (green) staining of p7 and ALP98 (red) staining of E2, and the staining showedstrong colocalization (yellow). Immunoblots of 293T cells expressing E1-E2-p7 via baculovirus transduction (see Materials and Methods) orE1-E2-SPm-p7 using ALP98 (10% Tris-glycine, top gel) and antibody 1055 (12.5% Tris-glycine, bottom gel [dark exposure to show uncleavedE2-p7 in the wild type]) showed the clear abrogation of signal peptidase-mediated cleavage of p7 from E2 in the SPm background.



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5) caused p7 to migrate significantly more slowly than unmod-ified p7 (lane 2) on both types of gel. This was most notable forFLAG-tagged p7 (FLAG-p7). In contrast, the presence of aC-terminal FLAG tag did not alter migration (lane 4). Fur-thermore, prolonged exposures of p7-FLAG lysates on Tris-glycine gels (Fig. 1B, bottom two blots, lane 3) revealed thatthe protein in fact migrates as two bands; the larger band ismore reactive to antibody 1055, and the smaller band is moredetectable with anti-FLAG. The bands could not be due todegradation of the protein, as they were both recognized bytwo antibodies, the epitopes for which were present at oppositetermini of the protein. This would also argue against the ex-pressed p7 being cleaved intracellularly at the cytosolic loop.The fact that the C-terminally tagged p7-FLAG did not appearto migrate differently than native p7 suggested that this doubletcould be a feature unique to modifying the p7 N terminus; thisfeature could potentially be due to a change in the overallcharge of the protein upon incorporation of a highly basic tagor perhaps due to a posttranslational modification or bindingof a cellular factor. Two forms could not be discerned forMYC-tagged p7 (MYC-p7), as this protein migrated closer tothe size of native p7. p7 expressed with its upstream signalpeptide, SP-p7, appeared to migrate as the native protein did,although in light of the migration of p7-FLAG, this does notnecessarily indicate the efficiency of signal peptide cleavage. p7with a mutated signal peptidase recognition site, SPm-p7, did,however migrate as a species that was visibly larger than thenative protein on Tris-glycine gels, yet due to the fact that itsmigration was akin to that of native p7 on MOPS gels, theeffect of the mutation on signal peptidase activity was assessedby alternate means (Fig. 2C).

Staining p7 with an antibody directed to its C terminusshows ER localization independent of the signal peptide. As aresult of inefficient signal peptidase-mediated cleavage, p7 hasbeen shown to exist in cells both as the native protein and as anE2-p7 precursor (26, 30). The membranes of the endoplasmicreticulum are the initial site of production of these proteins,and others have detected p7 in an ER-like compartment inHepG2 cells (11). Conversely, we demonstrated that tagged p7lacking the signal peptide from E2 localized to a mitochondri-on-like compartment in transfected 293T cells, suggesting thatp7 might be actively targeted to this cellular organelle (20). Wereasoned that the lack of the E2-derived signal peptide mightresult in aberrant targeting of p7, as the newly translated pro-tein would not be cotranslationally inserted into the ER mem-brane. To investigate this targeting in more detail, we thereforegenerated p7 expression constructs containing either the wild-type signal peptide from E2 upstream of p7 (SP-p7) or con-structs where the signal peptidase recognition site had beenaltered to prevent cleavage (SPm-p7) by introducing mutationsat the �3, �1 loci relative to the junction of the two proteins,A743N and A745R. This double mutation is based on thatpreviously shown to abrogate E2-p7 cleavage and viral infec-tivity in bovine viral diarrhea virus (21). 293T cells were fixedin 4% paraformaldehyde 16 h posttransfection, permeabilized,and stained using antibody 1055 and a marker for the ER,Texas Red-conjugated concanavalin A (Molecular Probes)prior to analysis on a digital deconvolution microscope (Delta-Vision). Sixteen hours was chosen as the time point, as cell

viability was higher at this time than at the 20 h used in ourprevious study.

The ER distribution of p7, as detected by antibody 1055, wasnot affected by the presence of the signal peptide or an epitopetag and overlapped with the concanavalin A signal (Fig. 2A)but not with Mitotracker CMXros (data not shown) in allcases. The appearance did not vary between brightly labeled ordimly labeled cells. Thus, native or tagged p7 appears capableof targeting to the ER posttranslationally in the absence of thesignal peptide from E2, and the presence of epitope tags doesnot cause erroneous targeting. The only difference in distribu-tion of C-terminally stained protein occurred where the signalpeptide cleavage site had been mutated, SPm-p7 (Fig. 2B). Inthis case, only dimly labeled cells showed a normal ER-typedistribution, whereas brightly labeled cells showed accumula-tion of cytoplasmic aggregates of p7, which appear to comprise,at least in part, modified ER membranes as judged by con-canavalin A staining (Fig. 2B). To confirm that this mutationblocked signal peptidase cleavage in cells, we introduced it intoa construct expressing E1 through to p7, pCDNAE1-SPm-p7.The staining for p7 (antibody 1055) strongly overlapped withthat for E2 (ALP98) in transfected cells expressing this con-struct. Immunoblot analysis confirmed the lack of signal pep-tide cleavage: a band migrating more slowly than E2 was de-tected by both ALP98 and antibody 1055. However, in thewild-type sequence, the majority of the p7 was cleaved from E2(Fig. 2C).

Staining the N or C terminus of p7 defines separate intra-cellular pools. We had previously shown that the use of ananti-FLAG antibody to detect an N-terminally FLAG-taggedp7, p7-FLAG, revealed a significant overlap with a mitochon-drial marker (20). The localization pattern of p7-FLAG ob-tained with antibody 1055, however, was not consistent withthis pattern of fluorescence. This raised the possibility that twoseparate pools of p7 existed within the cell and that these twopools differ in both subcellular localization and accessibility ofthe N and C termini to antibody.

We therefore reexamined the staining pattern for this con-struct in 293T cells with the anti-FLAG antibody at the earliertime point to investigate any temporal effects on p7 localiza-tion. At 16 h posttransfection we still obtained a population ofbrightly labeled cells showing a ring-like staining pattern con-sistent with localization to mitochondria or adjacent mem-branes that did indeed overlap with Mitotracker CMXros (Fig.3A and C). We noted, however, that a population of dimlylabeled cells was also present at this time point showing an ERlocalization for p7-FLAG (Fig. 3A). Close inspection of themitochondrial pattern revealed the staining to localize pre-dominantly to the outer surfaces of these MitotrackerCMXros-stained organelles, resulting in a ring-like distribution(Fig. 3C, top panels). To control for an effect attributable tothe presence of the FLAG tag, we also analyzed a constructwith an N-terminal MYC tag, MYC-p7, which displayed thesame phenotype as the FLAG-tagged construct (data notshown). Quantification of the FLAG fluorescence for the cellpopulations exhibiting each staining pattern confirmed that thecells showing a mitochondrial distribution of p7-FLAG stainedwith a directly conjugated anti-FLAG monoclonal antibodyconjugated to FITC were, indeed, up to twice the intensity(measured as mean fluorescence intensity per pixel [see Ma-



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terials and Methods]) as those exhibiting an ER pattern, sug-gesting that the localization was directly related to the level ofexpression of the protein (Fig. 3B). This analysis is more re-vealing than visual inspection of individual fluorescence im-ages, which are automatically adjusted to optimize signal/noisefor better contrast by digital image capture systems. For thisreason, images showing both phenotypes are often overex-posed for the brighter mitochondrial distribution or show thedimmer fluorescence poorly (Fig. 3A).

To reconcile this difference in labeling patterns, cells ex-pressing FLAG- or MYC-tagged constructs (data not shown)were dual labeled with both antibody 1055 and the antibody tothe epitope tag (Fig. 4). In both cases, brightly labeled cellsshowed little or no overlap between the ring-like N-terminalstaining and the C-terminal ER pattern. At lower levels, how-ever, there was an almost complete overlap (Fig. 4A). Tocontrol for the possibility that the epitope tags were cleaving ortargeting the protein nonspecifically to intracellular mem-branes, a construct in which the FLAG tag was located at theC terminus, p7-FLAG, was examined. This construct displayed

an ER-type distribution at all levels of labeling with eitherantibody; dual-labeled cells showed a complete overlap (Fig.4B). In all cases, staining with antibody 1055 showed an ERdistribution irrespective of the fluorescence intensity. Thus, theapparent difference in localization patterns was likely due todifferences in the accessibility of the p7 termini to antibodywhen p7 was present in different intracellular compartments.This masking effect could not be removed by permeabilizationin 0.1% SDS (data not shown).

To investigate whether this phenomenon occurred when p7was expressed with its signal peptide, constructs with an N-terminal epitope tag immediately downstream of the signalpeptide or with five amino acids inserted into the p7 sequence(to preserve the native cleavage site) were generated, as well asversions containing the K778A R780A mutations to reducepossible toxicity. Unfortunately, upon transfection into 293Tcells, these constructs gave similar phenotypes to that ofSPm-p7 with no evidence of ring-like staining. Western blotanalysis did show these constructs migrating as doublets,

FIG. 3. Staining the N terminus of p7 reveals two separate phenotypes in localization. (A) Staining the N terminus of p7-FLAG using ananti-FLAG antibody (�-FLAG) conjugated to FITC reveals two populations of cellular staining phenotypes; more brightly stained cells show aring-like staining pattern, whereas less brightly stained cells show a more ER-type distribution. The same pattern was observed for cells expressingMYC-p7 stained at the N terminus with 9E10 monoclonal antibody (data not shown). (B) To quantify the apparent difference in staining intensitiesobserved for cells exhibiting the ring-like, mitochondrial p7-FLAG staining pattern (mito) versus the less-intense ER-type distribution (er), 0.2-�mz sections of p7-FLAG-expressing cells stained with FITC-conjugated anti-FLAG antibody were taken at equal exposure and neutral densitysettings. Individual color channels were defined and subsequently quantified using the Image J program giving a value for mean fluorescenceintensity per pixel. The values are the means � standard errors of the means (error bars) for seven data sets. (C) To confirm that the two patternsof p7-FLAG stained with anti-FLAG antibody (�-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 (seeMaterials and Methods). Brightly staining cells (green) showed a clear overlap with the Mitotracker CMXros label (red) and that appeared toconcentrate in a ring around the outside of the organelles (top Merge panels), whereas the less bright staining overlapped strongly with theconcanavalin A signal (red, bottom Merge panels) and vice versa. Tx red, Texas red.



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though we could not confirm this was due to inefficient signalpeptide cleavage or to the N-terminal FLAG tag (data notshown). To address this issue, we are currently trying again togenerate an antibody to the p7 N terminus.

p7 is enriched in heavy membrane fractions and cofraction-ates with both normal ER and membranes associated withmitochondria. Antibody 1055 also allowed us to investigate p7localization by Western blotting of fractionated cellular ho-mogenates. Initially, clarified homogenates from transfected293T cells were subjected to a crude separation comparing amitochondrially enriched heavy membrane fraction, pelleted at10,000 � g, to light ER-derived microsomes pelleted at 100,000� g (Fig. 5A). Samples were blotted in parallel for proteinmarkers of the ER (calreticulin) and an inner mitochondrialmembrane protein (cytochrome oxidase subunit 1). As evidentfrom Fig. 5A, p7 was enriched in the heavy membrane fractionscompared to microsomes whether or not it retained its up-stream signal peptide (compare H and L lanes for p7 to H andL lanes for SP-p7). The presence of an epitope tag was alsoinconsequential, though again p7-FLAG appeared to migratemore slowly than p7-FLAG or native p7; the smaller p7-FLAGband was not visible on this exposure (Fig. 5A, H and L lanesfor Fp7). The enrichment of the p7-FLAG (H and L lanes forp7-F) protein in the heavy membranes was more apparent onreduced exposures of the gel (data not shown). SPm-p7 againmigrated more slowly than did p7 (though not at the same rateas p7-FLAG) in these experiments. SPm-p7 was also entirelypresent in the heavy membrane fraction, consistent with theformation of aggregates seen by fluorescence. This crude tech-nique does not separate mitochondria from their associatedER-derived membranes; hence, both heavy and light mem-brane fractions contained an approximately equal level of cal-reticulin, whereas only the heavy fractions contained cyto-chrome oxidase.

Next we examined the effects of expressing the other struc-tural proteins of HCV by transducing 293T cells with recom-binant baculoviruses expressing either Core-E1-E2-p7 or E1-E2-p7 (Fig. 5B). Again, p7 and to a more variable extent,E2-p7 were found to be enriched in the heavy membranefraction. E2 showed an approximately equal distribution be-tween the fractions, yet core protein was also enriched in theheavy membrane fraction; consistent with the recent reportthat it too has the ability to localize to mitochondria (39).

To determine whether the apparent mitochondrial localiza-tion of p7 was due to its presence in the organelle itself or inassociated ER-derived membranes, mitochondrially enrichedfractions were subjected to further purification by pelletingthrough a sucrose cushion followed by separation on a contin-uous sucrose gradient, a protocol modified from the method ofSchwer et al. (39). Mitochondria were tracked through theprocess via fluorimetric detection of Mitotracker CMXros withwhich the cells had been labeled prior to harvesting (data notshown). p7 in 293T cells transduced with a baculovirus express-ing Core-E1-E2-p7 was not found in purified mitochondria,but it was predominant in the associated ER-derived mem-branes (Fig. 5C). This membrane fraction remained in suspen-sion following the initial 5,000 � g spin that removes themajority of the mitochondria (Fig. 5C, lanes 2), but it subse-quently pelleted at 10,000 � g (lanes 3). In addition, a signif-icant proportion of p7 was found in microsomes pelleted at100,000 � g, consistent with an ER-resident pool (lanes 4).Despite a low level of calreticulin indicating traces of ERmembrane in the purified mitochondrial fractions (lanes 5 and6), no p7 was detectable in these samples. The relative levels of

FIG. 4. Separate pools of p7 defined by staining the N or C termi-nus of p7 protein. 293T cells transfected with p7-FLAG were duallabeled using C-terminal antibody 1055 (#1055) detected by an AlexaFluor 594-conjugated antibody and anti-FLAG antibody (�-FLAG)conjugated to FITC. The same results were obtained again forMYC-p7 stained using anti-MYC monoclonal antibody 9E10 detectedby a FITC-conjugated goat anti-mouse secondary antibody (data notshown). (A) p7-FLAG-expressing, bright FITC-labeled cells (green,Merge panel) showed a different staining pattern than the patternobtained for antibody 1055 (red), and these signals did not significantlyoverlap. Dim FITC-labeled cells, however, showed colocalization be-tween the signals for the two termini (yellow, Merge panel). (B) Tocontrol for artifacts arising from the use of the epitope tags, the samestaining protocol was performed on cells expressing pCDNAp7FLAGwhere the FLAG tag is located at the C terminus. In this case, thesignals from both antibodies overlapped independently of the FITClabeling intensity, indicating that the differences observed for p7-FLAG were likely due to differences in the accessibility of the proteintermini to the antibody.



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cytochrome oxidase to calreticulin combined with the very highsensitivity of the calreticulin antibody can be taken as meaningthat the overall level of ER membrane in these fractions wasminute; lanes 2, 5, and 6 displayed a significant enrichment for

mitochondrial proteins. E2-p7 was also present mainly in the10,000 � g pellet, though the levels of this species were lowoverall in these experiments. This would seemingly argueagainst cleavage of the signal peptide directing transport of p7

FIG. 5. p7 is enriched in heavy membrane fractions associated with mitochondria. Subcellular fractionation was performed to determine theproportion of p7 in ER- or mitochondrion-associated membrane fractions. (A) 293T cells transfected with various p7 expression constructs werehomogenized, and the membranes were separated by differential centrifugation (see Materials and Methods). Pellets obtained at 10,000 � g weredesignated heavy (H) and contained mitochondria along with associated ER-derived membranes, whereas those obtained at 100,000 � g weredesignated light (L) ER-derived microsomes. Normalized protein samples from each pellet were then subjected to SDS-PAGE and Westernblotting using antibody 1055 (#1055) against p7, the ER marker calreticulin (Cal), or the inner mitochondrial membrane marker cytochromeoxidase subunit 1 (COX). Abbreviations: p7, pCDNAp7; SP-p7, pCDNASP-p7; Fp7, pCDNAFLAG-p7; p7F, pCDNAp7-FLAG; SPmp7, pCD-NASPm-p7. (B) Heavy and light membrane fractions were prepared as described above for panel A from 293T cells transduced with baculovirusesexpressing 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 theseorganelles, 293T cells transduced with baculovirus expressing C-E1-E2-p7 were labeled with Mitotracker CMXros prior to homogenization. Crudemembrane fractions were generated by differential centrifugation (see Materials and Methods). The 5,000 � g pellet was then pelleted througha sucrose cushion, and mitochondria were purified on a continuous sucrose gradient as judged by Mitotracker CMXros fluorescence (data notshown). Protein content was normalized for all fractions, and samples were subjected to SDS-PAGE and Western blotting as in panel B. Lanes1, 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 peakmitochondrial fractions; lanes 7, supernatant after 100,000 � g spin, methanol precipitation, and pelleting (supernatant contains cytosol andresidual membranes).



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to membranes around mitochondria; however, it is likely thatthe 10,000 � g pellet contains other membrane compartmentsas well as those responsible for the mitochondrial localizationphenotype. In support of this argument, indirect fluorescencewith ALP98 (anti-E2) in 293T or HepG2 cells transduced withthe same baculovirus showed no mitochondrial labeling (Fig.1B and data not shown). Interestingly, very little core proteinwas present in gradient-purified mitochondria (requiring along exposure to be visualized; Fig. 5C, lanes 5 and 6) and whatthere was appeared to be p21 rather than fully processed p19,though the relevance of this observation is not clear. We con-clude that p7 is therefore present in both normal ER and alsoin a subset of ER closely associated with the mitochondrialouter membrane, potentially ER cisternae wrapped around theorganelle.

DISCUSSION

Our data are consistent with there being two distinct popu-lations of p7 distinguished by the accessibility of either the N orC terminus. p7 detected using antibodies to its C terminus isexclusively ER localized, whereas p7 detected with an N-ter-minal tag can either show an ER or an apparent mitochondriallocalization, depending on the level of labeling and presum-ably, therefore, of expression. This work provides evidence thatthe trafficking of HCV p7 is a complex process potentiallyregulated by both the cleavage from its upstream signal pep-tide and targeting signals present within the protein sequence.

It appears that many HCV genotypes show an inefficientcleavage of the E2-p7 precursor protein relative to the otherstructural proteins. It has recently been suggested that this mayregulate the generation of native p7 and so the formation ofion channel complexes and may provide a means of includingp7 in virions, a notion with which we are in complete agree-ment (10). Interestingly, signal peptide cleavage between E2and p7 in our baculovirus system appeared for the most part tobe reasonably efficient. This may be due to our using an HCVgenotype 1b protein which has been shown to cleave moreefficiently than HCV genotype 1a sequences (24). Further-more, in contrast to the other HCV structural proteins, thesignal peptidase responsible for the E2-p7 cleavage appears tobe unique to mammalian cells and is absent from insect cellsystems, suggesting that the virus may have evolved to regulatethis cleavage event via this specific pathway to achieve specificE2/E2-p7 ratios.

The development of an antibody to the C terminus of p7 hasallowed visualization of native protein for the first time. Wefound p7 to be present in the ER using this antibody regardlessof the presence or absence of the upstream signal peptide. Inthis regard, p7 exhibits targeting similar to that exhibited byHCV NS2, which has been shown to target the ER in theabsence of its upstream signal peptide, the C-terminal helix ofp7 (44). Labeling the N terminus, however, revealed a changein localization from an ER distribution in dimly labeled cells tomitochondrially adjacent membranes in brightly labeled cells.Assuming that the level of labeling directly correlates with thelevel of protein present, epitope-tagged p7 appears to movefrom the ER to membranes around mitochondria as labeling/expression increases. Transport is not complete, however, asC-terminally labeled protein is still detectable in the ER and

this signal does not overlap with that of the N-terminally la-beled protein. This implies that the pools of p7 protein in thesecompartments differ in the extent to which their termini areaccessible to antibody postfixation. Folding of the proteinwithin an oligomeric structure or a possible interaction with acellular factor may explain this observation, causing occlusionof the epitope. As this process appears to occur independentlyof the signal peptide from E2, its determinants must presum-ably reside within the p7 amino acid sequence. The C-terminalhelix of p7 has been shown to be capable of acting as a signalpeptide when fused to the HCV E1 protein, facilitating correctglycosylation of the protein in HepG2 cells (11). It is possiblethat the C-terminal helix of p7 may also direct in part thelocalization of the protein itself or be part of a multipartitesignal. It is becoming increasingly apparent that single trans-lation products can be targeted to distinct membrane compart-ments by competition between two internal signals, the out-come of which can be affected through a variety of mechanismsranging from protein folding to the metabolic status of the cell(reviewed in reference 25). Interestingly, p7 has been proposedto adopt two transmembrane topologies; its C terminus canpresent to either side of the ER membrane (24). It is possiblethat this process plays a role in or may be a consequence oftargeting of p7 to different membrane compartments.

Insertion of an epitope tag at the junction of the p7 Nterminus and its signal peptide appeared to interfere withprocessing; giving the same phenotype as the signal peptidecleavage site mutation did. Unfortunately, this made it impos-sible to tell by indirect fluorescence whether this transportevent occurs for native p7 after it has been targeted to the ERby its signal peptide. This is contrary to a recent report whereinsertion of a MYC tag near the p7 N terminus improvedcleavage efficiency for HCV genotype 1a p7 (10). That reportalso identified structural elements that appear to regulate pro-cessing at the E2-p7 junction. It is possible that disruption ofthese signals has different effects in separate virus genotypes;the termini of p7 have been shown to have a genotype-specificrole in chimeric HCV infectivity studies in chimpanzees inwhich the p7 protein of genotype 2a was introduced into ge-notype 1a background, as the only viable chimera was wherethe termini remained as genotype 1a sequence (38). In addi-tion, the genotype 1a and 1b p7 signal peptides appear tocleave with different efficiencies as determined by their aminoacid sequences (24). Our finding that mutation of the signalpeptidase recognition site disrupted the localization of p7 isconsistent with this proteolytic cleavage event playing an im-portant role in regulating p7 function. Having a kinetically slowcleavage might allow for direction of p7 into virions by retain-ing a pool in the ER as E2-p7, while cleaved protein passes tomitochondrially associated membranes, potentially forming ac-tive ion channel complexes. E2p7-MYC has been successfullydetected in VLPs made in insect cells by immunoelectronmicroscopy (24). It would be of interest to determine whetheruncleaved E2-p7 displays ion channel activity.

E2-p7 produced in the context of the other HCV structuralproteins via baculovirus transduction ought to retain authenticsignal peptidase processing. In this case, as well as by trans-fection, p7 was found to be enriched in the heavy membranefraction of 293T cell postnuclear homogenates which containalmost all the mitochondria as well as their associated ER-



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derived membranes. ER cisternae are known to wrap closelyaround mitochondria, facilitating rapid signaling and transportbetween the two organelles (42). In particular, the transmis-sion of calcium ion fluxes from ER cisternae to mitochondriahas been shown to be a pivotal process in the regulation ofapoptotic signaling (reviewed in reference 17). It is tempting tospeculate that the presence of p7 in these membranes mayinterfere with such signals, rendering the cell insensitive toproapoptotic stimuli from immune cells or the effects of otherviral gene products. The 2B protein of coxsackievirus B isstructurally quite similar to p7 and has recently been shown tohave an antiapoptotic effect via its influence on intracellularcalcium levels (9). Coincidentally, we found that p7 showedincreased in vitro ion channel activity with a calcium electrolytein planar lipid bilayers (19). Both 2B and p7 are purported tobelong to the same family of virus ion channels, viroporins.

Our analysis indicates that, as well as microsomes, p7 mostlikely associates with adjacent ER cisternae rather than withmitochondria per se; gradient-purified mitochondria containedno detectable p7, and the majority of the p7 protein remainedwith the 10,000 � g fraction after it had been substantiallydepleted of mitochondria by a 5,000 � g spin. The 10,000 � gpellet is also likely to contain specialized areas of ER mem-brane that directly contact mitochondrial membranes knowncollectively as mitochondrion-associated membranes, orMAMs (36, 42). These are biochemically distinct from bothnormal ER and mitochondrial membranes, being enriched inenzymes concerned with fatty acid metabolism, such as phos-phatidylserine synthase (41, 42). The HCV core protein hasalso recently been reported to localize to both mitochondria

and MAMs (39) following processing to p19 by signal peptidepeptidase in the ER, which also permits transport of the pro-tein to lipid droplets (29). We also detected core in purifiedmitochondria, though the protein appeared to migrate as un-processed p21 and it was present at low levels; its detectionrequired overexposure of relevant Western blots (Fig. 5C, mid-dle blot). Like p7, the majority of the p19 core protein ap-peared to reside in the cleared MAM fraction. It is possible,however, that some or all of the outer mitochondrial mem-brane had been removed during purification, resulting in theapparent absence of these proteins from the organelle, thoughthe presence of a low level of calreticulin in these purifiedfractions would argue against this. Nevertheless, the associa-tion of both core and p7 with mitochondria and/or associatedmembranes as well as the localization of core protein to lipiddroplets points to additional functions for these proteins in theHCV life cycle. HCV nonstructural proteins have also beenobserved to partially localize to ER cisternae around mito-chondria in replicon cells (31), and it is notable that ultrastruc-tural changes in mitochondria have been observed in chronicHCV patients (1).

As well as acting in membranes around mitochondria, thepool of p7 residing in the ER may also act in processes such asviral assembly. Pseudotyped retroviruses presenting HCV E1and E2 on their surface are known to show pH-dependent cellentry (22), likely due to E2 adopting a fusogenic conformationprematurely. It is conceivable that p7 may protect E2 fromsuch pH-induced changes during assembly/entry in the sameway that both it and M2 can protect influenza A virus hemag-glutinin (14, 20, 37).

FIG. 6. Hypothetical model for intracellular targeting of p7. Upon translation, p7 adopts either a single- or double-membrane-spanningtopology. 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 ofthe protein. If signal peptide cleavage occurs, double-membrane-spanning p7 will form oligomeric channels, whereas uncleaved E2-p7 is directedinto virus particles. A single-membrane-spanning p7 (bottom left) could spontaneously adopt the double-membrane-spanning topology prior tosignal peptide cleavage, or by the action of a C-terminal signal, cleaved protein could be targeted to membranes around mitochondria. Uponreaching these membranes, protein would then be free to adopt a double-membrane-spanning topology forming oligomeric channels.



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It is possible to construct a model for the regulation of p7localization by combining the role of the signal peptide, itstransmembrane topology, and the presence of internal signalsequences (Fig. 6). Upon translation in the rough ER, a pro-portion of p7 remains with its C-terminal helix on the cytosolicside of the membrane. As levels of protein increase, a potentialsignal in the C terminus of the protein binds to a cellular factorthat then directs this population of protein to membranesaround mitochondria. Conversely, the remaining protein withboth termini on the luminal side of the membrane is bound bya factor that causes ER retention. Signal peptide cleavagecould theoretically occur in any of the ER-derived membranes,though it is perhaps delayed by binding of an ER retentionfactor such that a pool of E2-p7 remains in the ER for incor-poration into virions. If signal peptide cleavage occurs in themitochondrial ER cisternae, E2 might be channelled back tothe rough ER, whereas p7 remains and adopts its dual-span-ning topology. Cleaved p7 in both sets of membranes wouldthen be free to oligomerize and form ion channels in eithermembrane. In addition, p7 incorporated into virions as E2-p7may also be processed during exocytosis to allow the formationof channels that protect E2 from fusogenic change and/orfunction during virus entry. The recently available HCV rep-lication systems based on the HCV genotype 2a JFH-1 strainthat produces infectious virus particles would be an ideal sys-tem in which to determine any potential role for p7 in virusexit/entry. Unfortunately, however, antibody 1055 will not beemployable as a tool in this regard due to differences in the Ctermini of p7 proteins of genotype 1b and 2a virus isolates. Weare, however, pursuing this line of investigation with a view todeveloping new anti-p7 antibodies specific to other HCV ge-notypes.

As more insights into p7 function and behavior in cells aregained, it is clear that targeting p7 in future antiviral therapiescould potentially act by blocking HCV at multiple points in itslife cycle, perhaps using compounds based on amantadine de-rivatives. Further experiments on ion channel function, ideallyin systems where HCV virions can be produced, will be re-quired to define the precise function of p7 in HCV replication.

ACKNOWLEDGMENTS

We thank Helen Bright and Tony Carroll (Glaxo-Smith-Kline,Stevenage, United Kingdom) for help in developing antibody 1055. Wealso thank Andrew Street (University of Leeds) for the NS5A-GFPconstruct, as well as Matthew Bentham and Gareth Howell (Universityof Leeds) for useful discussions. Fluorescence microscopy was under-taken in the Wellcome Trust Bio-imaging facility in the Faculty ofBiological Sciences of the University of Leeds.

This work was supported by grants from the Wellcome Trust(067125 and 074023). Dean Clarke was supported by a Medical Re-search Council Ph.D. studentship.

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