1 INSERM, U412, Lyon, F-69007 France
2 Ecole Normale Supérieure de Lyon, Lyon, F-69007 France
3 IFR128 BioSciences Lyon-Gerland, Lyon, F-69007 France
4 Laboratoire de Virologie et Pathogénèse Virale, CNRS UMR-5537, Faculté de Médecine de Lyon and Institut Fédératif de Recherche RTH Laennec, Lyon, France
5 Institut de Biologie et Chimie des Proteines, CNRS-UMR 5086, Université Claude Bernard Lyon 1, Lyon, France
Correspondence
François-Loïc Cosset
flcosset{at}ens-lyon.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Published online ahead of print on 6 October 2005 as DOI 10.1099/vir.0.81428-0.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The HCV envelope GPs expressed in heterologous systems were shown to be retained at the ER membrane by retention signals, including charged residues in the middle of their transmembrane domains (Cocquerel et al., 1998, 1999
; Flint & McKeating, 1999
). Mutation of these charged residues has been shown to abolish ER retention, but also to interfere with heterodimerization of E1 and E2 (Cocquerel et al., 2000
; Michalak et al., 1997
; Op De Beeck et al., 2000
; Patel et al., 2001
). Indeed, the transmembrane domains of E1 and E2 play a major role in the assembly of E1 and E2 into non-covalently attached heterodimers, which are thought to be the prebudding form of the HCV GPs (Deleersnyder et al., 1997
; Dubuisson, 2000
).
Since its discovery 16 years ago, HCV has been difficult to study because it does not replicate efficiently or form particles in vitro. To establish surrogate model systems for HCV particle production, several laboratories initially tried to develop virus-like particles or pseudotype viruses by, for example, incorporating the HCV GPs onto cores of heterologous viruses, including those of vesicular stomatitis virus (VSV) or influenza virus (Flint et al., 1999; Lagging et al., 1998
; Matsuura et al., 2001
; Takikawa et al., 2000
). In such systems, assembly was thought to take place at the cell surface; therefore, the HCV GPs were retargeted away from the ER to the plasma membrane by mutation or replacement of their transmembrane domains to achieve relocalization and thus incorporation onto heterologous viral cores. Because assembly and functionality of HCV GPs are very sensitive to mutations and deletion within their transmembrane domains and because the HCV GPs have a tendency to misfold and aggregate (Cocquerel et al., 2000
; Dubuisson, 2000
; Dubuisson et al., 2000
), these attempts were mainly unsuccessful (Buonocore et al., 2002
). Recently, production methods for replication-competent HCV particles in vitro (HCVcc) have been reported; however, they are restricted to safety-level 3 laboratories (Lindenbach et al., 2005
; Wakita et al., 2005
; Zhong et al., 2005
).
We and others have recently described HCV pseudoparticles (HCVpp) that are assembled by incorporating unmodified, full-length HCV GPs onto oncoretroviral or lentiviral cores (Bartosch et al., 2003b; Drummer et al., 2003
; Hsu et al., 2003
) that are highly infectious and that seem to mimic the viral entry and serological properties of wild-type HCV (Bartosch et al., 2003a
, c
; Logvinoff et al., 2004
). HCVpp can be produced in large quantities at comparatively high titres and at a convenient safety level. Furthermore, they offer great flexibility in terms of incorporation of marker genes and allow investigation of viral entry independently of replication, as attachment and fusion are mediated by the HCV GPs and post-fusion steps are mediated by retro- or lentiviral core particles. Due to these features, HCVpp are likely to remain a valuable tool that will complement studies with the wild-type virus.
HCVpp are produced by expressing the E1E2 glycoproteins, the retroviral core proteins and a packaging-competent retroviral genome carrying a marker gene in human 293T cells (Bartosch et al., 2003b). Viruses assembled by the 293T producer cells are collected from the supernatant and used to infect naive target cells. Within the 293T producer cells, E1 and E2 are expressed mainly at the ER, but a small fraction traffics to the cell surface (Bartosch et al., 2003b
; Drummer et al., 2003
; Hsu et al., 2003
). Recent insights into retroviral assembly show that assembly and incorporation of diverse viral GPs may not take place at the cell surface, but can occur intracellularly within the endocytic pathway (Nydegger et al., 2003
; Pelchen-Matthews et al., 2003
; Sherer et al., 2003
). In particular, budding of human immunodeficiency virus and Murine leukemia virus (MLV) has been shown to occur into multivesicular bodies (MVBs), a late endosomal compartment that can fuse with the cell surface (Gould et al., 2003
). We therefore investigated here the cellular site of assembly of MLV-based HCVpp. By using a combination of biochemical methods and confocal and electron microscopy (EM) to reveal the cellular localization of expressed E1 and E2 GPs, as well as MLV core proteins, we show that HCVpp bud intracellularly, presumably into MVBs. In addition, by studying the mutual requirement of E1 and E2 for the formation of infectious particles, we found that E2 can be incorporated efficiently onto retroviral core particles in the absence of E1, whilst the incorporation of E1 onto HCVpp is strongly dependent on the presence of E2.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies.
The mAb p5D4 (Sigma-Aldrich), against VSV-G, was used diluted to 1 : 10 000 for Western blotting and to 1 : 2000 for immunofluorescence (IF) experiments. Anti-RD114 GP (ViroMed Biosafety Labs), a goat antiserum raised against the RD114 gp70 envelope surface protein (SU), was used at 1 : 3000 for IF experiments. The HCV-E2 GP was detected with mouse H52 at 1 : 1000 for Western blotting and with undiluted H53 hybridoma supernatant for IF studies. The HCV-E1 GP was detected with mouse A4 at 1 : 1000 for Western blotting and at 1 : 500 for IF studies. Anti-MLV capsid (MLV CA; ViroMed Biosafety Labs) is a goat antiserum raised against the Rauscher leukemia virus p30 capsid and was used at 1 : 10 000 dilution for Western blotting. A rabbit antiserum against MLV capsid p30 (a gift from A. Rein, National Cancer Institute, Frederick, MD, USA) was used at 1 : 10 000 dilution to identify MLV Gag in IF studies. The secondary Alexa antibodies used for IF were purchased from Molecular Probes.
Production of HCVpp and infection assays.
Production of HCVpp and infection assays have been described previously (Bartosch et al., 2003b). To analyse the incorporation of HCV envelope GPs into pseudoparticles, HCVpp were pelleted by centrifugation through 20 % sucrose cushions and analysed by Western blotting.
Biotinylation and Western blotting.
Forty hours post-transfection, virion-producer cells were chilled on ice, washed twice with cold PBS (pH 8·0) supplemented with 0·7 mM CaCl2 and 0·25 mM MgSO4 (PBS++) and incubated with 0·5 mg sulfo-NHS-LC-LC-biotin ml1 (Pierce) for 30 min at 4 °C. Biotinylation was stopped by incubating the cells with 1 M glycine in PBS++ for 5 min at 4 °C. The cells were then washed with PBS/0·1 M glycine, lysed with MacDougal buffer [20 mM Tris/HCl (pH 8·0), 120 mM NaCl, 200 µM EGTA, 0·2 µM NaF, 0·2 % sodium deoxycholate, 0·5 % Nonidet P-40] containing a protease-inhibitor cocktail (Complete Mini; Roche Diagnostics) and 0·1 M glycine, and centrifuged at 13 000 g for 30 min; 80 % of the cell lysates were incubated overnight at 4 °C with streptavidinSepharose beads (Pierce). The beads were then washed with MacDougal glycine buffer, resuspended in a denaturing buffer (1 % -mercaptoethanol, 0·5 % SDS) and boiled for 5 min. Purified virus samples were obtained by ultracentrifugation of viral supernatants through a 1·5 ml 20 % sucrose cushion in a Beckman SW41 rotor (25 000 r.p.m., 2·5 h, 4 °C) and suspended in PBS. All samples were mixed 5 : 1 (v/v) with a loading buffer [375 mM Tris/HCl (pH 6·8) containing 6 % SDS, 30 %
-mercaptoethanol, 10 % glycerol and 0·06 % bromophenol blue], boiled for 5 min and then analysed by SDS-PAGE (12 % gel). Western blotting was performed by using standard procedures. SuperSignal West Pico chemiluminescent substrate (Pierce) was used to reveal proteins.
IF and confocal microscopy imaging.
FuGENE 6 (Roche Diagnostics)-transfected virus-producer cells were grown on 35 mm diameter coverglass dishes coated with D-lysine (Mattek Corporation) or on uncoated 14 mm diameter glass coverslips. IF staining was performed at room temperature 40 h post-transfection. The cells were washed with PBS, fixed for 15 min in 3 % paraformaldehyde/PBS, quenched with 50 mM NH4Cl and permeabilized in 0·2 % Triton X-100 for 8 min. Fixed cells were incubated for 1 h with primary antibody in 1 % BSA/PBS, washed and stained for 1 h with the corresponding fluorescent, Alexa-conjugated secondary antibody (at 0·5 µg ml1) in 1 % BSA/PBS. The cells were then washed several times with PBS and mounted on microscope slides with the antifading agent Prolong (Molecular Probes). Images were acquired with an LSM 510 confocal microscope equipped with an Axiovert 100 M microscope (Carl Zeiss) and a 63x1·3 numerical aperture Apocromat objective. Alexa 488 was excited with an argon laser line at 488 nm and emissions were collected with a band-pass filter (BP505550). Alexa 546 or 555 was excited, independently of Alexa 488, with a HeNe laser line at 543 nm and emissions were collected with a long-pass filter (LP560).
EM.
293T virion-producer cells were harvested 48 h after transfection, pelleted, fixed with 2 % glutaraldehyde in 0·1 M sodium cacodylate buffer (pH 7·4) and post-fixed with osmium tetroxide [1 % in 0·1 M cacodylate buffer (pH 7·4)]. Cell specimens were dehydrated and embedded in Epon (Epon-812; Fulham). Sections were stained with 7 % uranyl acetate in methanol and post-stained with 2·6 % alkaline lead citrate in H2O. Specimens were examined under a JEOL 1200-EX electron microscope and analysed with a MegaView II high-resolution TEM camera and the Soft Imaging system (Eloïse). For quantitive EM analysis, at least 50 different cell sections were examined and pseudoparticles were counted in the cytoplasm, in MVBs and at the plasma membrane. For determination of particle size and sphericity index (Gay et al., 1998), a mean diameter for each individual particle was obtained by averaging at least three different diameters measured at 60° angles on the circle delineated by each sectioned particle. To establish the diameter of particles in a given cellular compartment, a minimum of 20 particles were measured.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Finally, we also observed particles within the cytoplasm of HCVpp-producing cells (Fig. 3b; Table 1
). These intracellular particles, however, had a small diameter (approx. 82·52±12·74 nm) when compared with particles produced from cells expressing the MLV core proteins alone (91·94±7·7 nm) (Table 1
). They may represent non-enveloped, cytoplasmic core particles. Cells producing VSV-Gpp did not contain particles within MVBs or the cytoplasm, suggesting that these features are specific to HCVpp-producing cells (Table 1
). Thus, to determine which HCV GP induced these phenomena, we investigated by EM the distribution of particles in cells expressing E1 or E2 individually with MLV core proteins. In cells co-expressing E2 and MLV core proteins, particles were observed at the cell surface and in MVBs, but not within the cytoplasm (Table 2
). In contrast, in cells co-expressing E1 and MLV core proteins, the proportion of cytoplasmic particles had increased by approximately twofold (from 22 to 41 %; Table 2
) compared with cells expressing both E1E2 GPs and MLV core proteins. Furthermore, by using confocal microscopy, whilst we detected some colocalization between MLV core proteins and E2 expressed in the absence of E1 (Fig. 4
ac), we did not detect any colocalization between MLV core proteins and E1 expressed in the absence of E2 (Fig. 4d
f).
|
|
Intracellular forms of E2 are incorporated preferentially onto MLV core particles
To confirm and extend our microscopic observations on intracellular HCVpp assembly, we performed biotinylation studies of E1 and E2. Comparison of the amounts and electrophoretic mobilities of biotinylated E1 and E2 expressed at the cell surface to E1 and E2 present on viral particles or in total cell lysates by immunoblotting is shown in Fig. 5. Specificity of biotinylation for cell surface-expressed proteins was controlled by detection of the retroviral core proteins, which are protected from biotinylation by either cell or viral membranes (Fig. 5
, bottom panels). Examination of the electrophoretic mobility of E2 revealed different isoforms on viral particles (Fig. 5b
, right panel). Importantly, the E2 species found at the cell surface of HCVpp-producing cells migrated much faster in denaturing reducing SDS-PAGE than E2 species incorporated on virions. Because virion-associated E2 species are rather heterogeneous and sensitive to peptide : N-glycosidase F digestion (data not shown) (Op De Beeck et al., 2004
) whilst cell surface-expressed E2 species migrate with much higher mobility, E2 proteins must be incorporated into viral particles intracellularly at a stage before the trimming process is complete. After incorporation into viral particles, E2 must be protected from further trimming, whereas unincorporated, monomeric E2 protein is subject to further trimming before it finally reaches the cell surface. These biochemical data, suggesting intracellular recruitment of E2, are therefore fully consistent with our microscopic observations. In contrast, no clear variation of the electrophoretic mobility of E1 incorporated onto viral particles was observed when compared to E1 monomer in total cell lysate or on the cell surface of HCVpp-producing cells (Fig. 5b
, left panel).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Determination of structure and assembly of wild-type HCV in vivo remains a challenging issue. Reported data based on HCV viral-like particles and HCV replicons suggest that replication occurs in ER-derived compartments and HCV budding may be driven by the core protein (Baumert et al., 1998; Blanchard et al., 2002
, 2003
; Egger et al., 2002
). However, the cellular site of wild-type HCV assembly has remained elusive so far and the close association of HCV biology with lipoprotein metabolism further complicates current views on HCV morphogenesis (André et al., 2005
). Previous EM studies on HCV structure have shown that the virus measures 5060 nm in diameter (Kaito et al., 1994
; Shimizu et al., 1996
). Wild-type HCV is thus considerably smaller than HCVpp (which has a diameter of 104 nm), a size difference that is probably due to the core proteins. MLV core particles devoid of viral GPs measure 92 nm, whilst the size of HCV nucleocapsid-like particles has been reported in the range 3862 nm (André et al., 2005
). Regarding the assembly of wild-type HCV, Shimizu et al. (1996)
have reported the detection of enveloped particles in cytoplasmic vesicles of HCV-replicating cells, which suggested that the morphogenesis of wild-type HCV may be vesicle-orientated. Interestingly, they detected HCV in cytoplasmic vesicles that potentially resemble MVBs. With the very recent development of systems that support wild-type HCV production in vitro (Lindenbach et al., 2005
; Wakita et al., 2005
; Zhong et al., 2005
), it will be interesting to see whether an involvement of MVBs in wild-type HCV morphogenesis can be confirmed.
The respective roles of E1 and E2 in particle assembly were clarified in this study by investigating the expression patterns of the HCV GPs E1 and/or E2 expressed individually with MLV core proteins. In cells expressing E2 with MLV core proteins, most particles were observed at the cell surface, suggesting efficient particle assembly and egress. In contrast, in cells co-expressing E1 with MLV core proteins, an accumulation of non-enveloped particles in the cytoplasm was observed, suggesting that E1 may possibly inhibit particle assembly and egress by an unknown mechanism. Because the inhibition of E1 on particle assembly and/or egress can be overcome by co-expression of E2, and because E1 colocalizes efficiently with retroviral core only in the presence of E2, our findings suggest that E1 incorporation onto pseudoparticles occurs subsequent to E1E2 heterodimerization. This finding is consistent with previous studies, which showed that the prebudding form of E1E2 is a heterodimer (Cocquerel et al., 2000; Michalak et al., 1997
; Op De Beeck et al., 2000
, 2004
; Patel et al., 2001
). Furthermore, we found a direct correlation between the presence of both E1 and E2 and the infectivity of HCVpp, indicating that heterodimer formation and functionality of E1 and E2 are tightly linked processes.
In conclusion, the assembly of HCV GPs on pseudoparticles may be more similar to that of wild-type HCV than currently thought. The morphogenesis of HCVpp requires both HCV GPs and does not occur at the cell surface, but rather is vesicle-orientated and leads to the formation of functional, fusogenic HCV GP complexes on the virion surface.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
André, P., Perlemuter, G., Budkowska, A., Bréchot, C. & Lotteau, V. (2005). Hepatitis C virus particles and lipoprotein metabolism. Semin Liver Dis 25, 93104.[CrossRef][Medline]
Bartosch, B., Bukh, J., Meunier, J.-C., Granier, C., Engle, R. E., Blackwelder, W. C., Emerson, S. U., Cosset, F.-L. & Purcell, R. H. (2003a). In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proc Natl Acad Sci U S A 100, 1419914204.
Bartosch, B., Dubuisson, J. & Cosset, F.-L. (2003b). Infectious hepatitis C virus pseudo-particles containing functional E1E2 envelope protein complexes. J Exp Med 197, 633642.
Bartosch, B., Vitelli, A., Granier, C. & 7 other authors (2003c). Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem 278, 4162441630.
Baumert, T. F., Ito, S., Wong, D. T. & Liang, T. J. (1998). Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J Virol 72, 38273836.
Blanchard, E., Brand, D., Trassard, S., Goudeau, A. & Roingeard, P. (2002). Hepatitis C virus-like particle morphogenesis. J Virol 76, 40734079.
Blanchard, E., Hourioux, C., Brand, D., Ait-Goughoulte, M., Moreau, A., Trassard, S., Sizaret, P.-Y., Dubois, F. & Roingeard, P. (2003). Hepatitis C virus-like particle budding: role of the core protein and importance of its Asp111. J Virol 77, 1013110138.
Buonocore, L., Blight, K. J., Rice, C. M. & Rose, J. K. (2002). Characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis C virus glycoproteins. J Virol 76, 68656872.
Cocquerel, L., Meunier, J.-C., Pillez, A., Wychowski, C. & Dubuisson, J. (1998). A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol 72, 21832191.
Cocquerel, L., Duvet, S., Meunier, J.-C., Pillez, A., Cacan, R., Wychowski, C. & Dubuisson, J. (1999). The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum. J Virol 73, 26412649.
Cocquerel, L., Wychowski, C., Minner, F., Penin, F. & Dubuisson, J. (2000). Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins. J Virol 74, 36233633.
Deleersnyder, V., Pillez, A., Wychowski, C., Blight, K., Xu, J., Hahn, Y. S., Rice, C. M. & Dubuisson, J. (1997). Formation of native hepatitis C virus glycoprotein complexes. J Virol 71, 697704.[Abstract]
Drummer, H. E., Maerz, A. & Poumbourios, P. (2003). Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins. FEBS Lett 546, 385390.[CrossRef][Medline]
Dubuisson, J. (2000). Folding, assembly and subcellular localization of hepatitis C virus glycoproteins. Curr Top Microbiol Immunol 242, 135148.[Medline]
Dubuisson, J., Duvet, S., Meunier, J.-C., Op De Beeck, A., Cacan, R., Wychowski, C. & Cocquerel, L. (2000). Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein. J Biol Chem 275, 3060530609.
Dubuisson, J., Penin, F. & Moradpour, D. (2002). Interaction of hepatitis C virus proteins with host cell membranes and lipids. Trends Cell Biol 12, 517523.[CrossRef][Medline]
Dumonceaux, J., Cormier, E. G., Kajumo, F., Donovan, G. P., Roy-Chowdhury, J., Fox, I. J., Gardner, J. P. & Dragic, T. (2003). Expression of unmodified hepatitis C virus envelope glycoprotein-coding sequences leads to cryptic intron excision and cell surface expression of E1/E2 heterodimers comprising full-length and partially deleted E1. J Virol 77, 1341813424.
Egger, D., Wölk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D. & Bienz, K. (2002). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76, 59745984.
Flint, M. & McKeating, J. A. (1999). The C-terminal region of the hepatitis C virus E1 glycoprotein confers localization within the endoplasmic reticulum. J Gen Virol 80, 19431947.
Flint, M., Thomas, J. M., Maidens, C. M., Shotton, C., Levy, S., Barclay, W. S. & McKeating, J. A. (1999). Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol 73, 67826790.
Galli, T., Chilcote, T., Mundigl, O., Binz, T., Niemann, H. & De Camilli, P. (1994). Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells. J Cell Biol 125, 10151024.[Abstract]
Gay, B., Tournier, J., Chazal, N., Carrière, C. & Boulanger, P. (1998). Morphopoietic determinants of HIV-1 Gag particles assembled in baculovirus-infected cells. Virology 247, 160169.[CrossRef][Medline]
Gosert, R., Egger, D., Lohmann, V., Bartenschlager, R., Blum, H. E., Bienz, K. & Moradpour, D. (2003). Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77, 54875492.
Gould, S. J., Booth, A. M. & Hildreth, J. E. K. (2003). The Trojan exosome hypothesis. Proc Natl Acad Sci U S A 100, 1059210597.
Greive, S. J., Webb, R. I., Mackenzie, J. M. & Gowans, E. J. (2002). Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection. J Viral Hepat 9, 917.[CrossRef][Medline]
Guibinga, G. H., Hall, F. L., Gordon, E. M., Ruoslahti, E. & Friedmann, T. (2004). Ligand-modified vesicular stomatitis virus glycoprotein displays a temperature-sensitive intracellular trafficking and virus assembly phenotype. Mol Ther 9, 7684.[Medline]
Hsu, M., Zhang, J., Flint, M., Logvinoff, C., Cheng-Mayer, C., Rice, C. M. & McKeating, J. A. (2003). Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci U S A 100, 72717276.
Kaito, M., Watanabe, S., Tsukiyama-Kohara, K. & 7 other authors (1994). Hepatitis C virus particle detected by immunoelectron microscopic study. J Gen Virol 75, 17551760.[Abstract]
Lagging, L. M., Meyer, K., Owens, R. J. & Ray, R. (1998). Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus. J Virol 72, 35393546.
Lindenbach, B. D. & Rice, C. M. (2001). Flaviviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 9911041. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Lindenbach, B. D., Evans, M. J., Syder, A. J. & 8 other authors (2005). Complete replication of hepatitis C virus in cell culture. Science 309, 623626.
Logvinoff, C., Major, M. E., Oldach, D. & 7 other authors (2004). Neutralizing antibody response during acute and chronic hepatitis C virus infection. Proc Natl Acad Sci U S A 101, 1014910154.
Mackenzie, J. M. & Westaway, E. G. (2001). Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol 75, 1078710799.
Major, M. E., Rehermann, B. & Feinstone, S. M. (2001). Hepatitis C viruses. In Fields Virology, 4th edn, pp. 11271161. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Matsuura, Y., Tani, H., Suzuki, K. & 8 other authors (2001). Characterization of pseudotype VSV possessing HCV envelope proteins. Virology 286, 263275.[CrossRef][Medline]
Michalak, J.-P., Wychowski, C., Choukhi, A., Meunier, J.-C., Ung, S., Rice, C. M. & Dubuisson, J. (1997). Characterization of truncated forms of hepatitis C virus glycoproteins. J Gen Virol 78, 22992306.[Abstract]
Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T. & Sato, J. (1982). Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 42, 38583863.[Abstract]
Nydegger, S., Foti, M., Derdowski, A., Spearman, P. & Thali, M. (2003). HIV-1 egress is gated through late endosomal membranes. Traffic 4, 902910.[CrossRef][Medline]
Op De Beeck, A., Montserret, R., Duvet, S., Cocquerel, L., Cacan, R., Barberot, B., Le Maire, M., Penin, F. & Dubuisson, J. (2000). The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization. J Biol Chem 275, 3142831437.
Op De Beeck, A., Cocquerel, L. & Dubuisson, J. (2001). Biogenesis of hepatitis C virus envelope glycoproteins. J Gen Virol 82, 25892595.
Op De Beeck, A., Voisset, C., Bartosch, B., Ciczora, Y., Cocquerel, L., Keck, Z., Foung, S., Cosset, F.-L. & Dubuisson, J. (2004). Characterization of functional hepatitis C virus envelope glycoproteins. J Virol 78, 29943002.
Orenstein, J. M., Meltzer, M. S., Phipps, T. & Gendelman, H. E. (1988). Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study. J Virol 62, 25782586.[Medline]
Patel, J., Patel, A. H. & McLauchlan, J. (2001). The transmembrane domain of the hepatitis C virus E2 glycoprotein is required for correct folding of the E1 glycoprotein and native complex formation. Virology 279, 5868.[CrossRef][Medline]
Pelchen-Matthews, A., Kramer, B. & Marsh, M. (2003). Infectious HIV-1 assembles in late endosomes in primary macrophages. J Cell Biol 162, 443455.
Penin, F., Dubuisson, J., Rey, F. A., Moradpour, D. & Pawlotsky, J.-M. (2004). Structural biology of hepatitis C virus. Hepatology 39, 519.[CrossRef][Medline]
Pornillos, O., Garrus, J. E. & Sundquist, W. I. (2002). Mechanisms of enveloped RNA virus budding. Trends Cell Biol 12, 569579.[CrossRef][Medline]
Raposo, G., Moore, M., Innes, D., Leijendekker, R., Leigh-Brown, A., Benaroch, P. & Geuze, H. (2002). Human macrophages accumulate HIV-1 particles in MHC II compartments. Traffic 3, 718729.[CrossRef][Medline]
Robertson, B., Myers, G., Howard, C. & 14 other authors (1998). Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. Arch Virol 143, 24932503.[CrossRef][Medline]
Sandrin, V., Muriaux, D., Darlix, J.-L. & Cosset, F.-L. (2004). Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses. J Virol 78, 71537164.
Sherer, N. M., Lehmann, M. J., Jimenez-Soto, L. F. & 7 other authors (2003). Visualization of retroviral replication in living cells reveals budding into multivesicular bodies. Traffic 4, 785801.[CrossRef][Medline]
Shimizu, Y. K., Feinstone, S. M., Kohara, M., Purcell, R. H. & Yoshikura, H. (1996). Hepatitis C virus: detection of intracellular virus particles by electron microscopy. Hepatology 23, 205209.[Medline]
Takikawa, S., Ishii, K., Aizaki, H., Suzuki, T., Asakura, H., Matsuura, Y. & Miyamura, T. (2000). Cell fusion activity of hepatitis C virus envelope proteins. J Virol 74, 50665074.
Wakita, T., Pietschmann, T., Kato, T. & 9 other authors (2005). Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11, 791796.[CrossRef][Medline]
Zhong, J., Gastaminza, P., Cheng, G. & 7 other authors (2005). Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102, 92949299.
Received 11 August 2005;
accepted 28 September 2005.