Infectious Disease Research Centre, Pav. CHUL, University of Laval, 2705 boulevard Laurier, Québec (Québec), Canada G1V 4G2
Correspondence
Denis Leclerc
Denis.Leclerc{at}crchul.ulaval.ca
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
hspp is a presenilin-related aspartic protease that catalyses intramembrane proteolysis of signal peptides. This protein comprises seven to nine putative transmembrane regions embedded in the ER (Golde & Younkin, 2001) and exists as a functional homodimer of 95 kDa (Nyborg et al., 2004
). The cleavage of C by hspp is believed to be important, as only the hspp-matured form of C is found in HCV virions isolated from the blood of infected patients (Yasui et al., 1998
). This maturation is also linked to the migration of C to the nucleus, lipid droplets (Barba et al., 1997
; McLauchlan et al., 2002
) and mitochondrion-associated ER membrane (MAM) (Schwer et al., 2004
). The association of C with lipid structures appears to be crucial for the prevention of degradation of the protein by proteasomes (Suzuki et al., 2001
).
Circulating HCV particles in chronically infected patients can be divided into different populations: (i) lipoprotein-associated virions with densities of 1·001·06 g ml1 on a sucrose gradient; (ii) free enveloped virions with a density of 1·081·11 g ml1; (iii) virus particles of 1·171·21 g ml1, associated with immunoglobulins; and (iv) non-enveloped particles with high densities of 1·221·25 g ml1 (Miyamoto et al., 1992; Hijikata et al., 1993
; Kanto et al., 1994
; Choo et al., 1995
; Prince et al., 1996
; Ishida et al., 2001
; André et al., 2002
). In chronic infections, the dominant population shifts from low-density to high-density particles with the progression of liver disease or inflammation (Kanto et al., 1995
; Watson et al., 1996
). A plausible explanation for the presence of non-enveloped particles in the blood is their release into the circulation by the lysis of infected hepatocytes that accompanies liver inflammation. Non-enveloped particles have also been detected as viral inclusions in the cytoplasm of liver cells of infected patients (Falcón et al., 2003
). However, the production of non-enveloped particles and their secretion are not very well understood.
Yeast has been shown to be useful in the study of virus replication and assembly (Sakuragi et al., 2002; Schwartz et al., 2004
). Expression of C in the yeast Pichia pastoris leads to the formation of nucleocapsid-like particles (NLPs) that are very similar in size and shape to the virus found in the blood of infected patients (Falcón et al., 1999
; Acosta-Rivero et al., 2001
). In this study, we investigated the effect of hspp cleavage on NLP assembly and formation in yeast. We observed the formation of NLPs in cells expressing C in the absence of maturation. Co-expression of hspp correlated with an increase in the number of non-enveloped particles. Interestingly, the hspp protein was present at the surface of the enveloped NLPs. It appears that the protein is captured together with the membranes during virion formation and is exposed at the surface of the virus. A model for HCV particle formation is discussed.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
C (1179) was amplified by PCR using primers 5'-CCTCCCATGGTGAGCACGAATCCTAAACCTCAAGAAAAAACC-3' and 5'-GACGGGATCCTCACAGAAGGAAGATAGAGAAAGAGCAACC-3', and cloned as an NcoIBamHI fragment in vector pET-3d (Novagen). Escherichia coli strain BL21 Star (Invitrogen) was then used to express the protein as described previously (Majeau et al., 2004).
Plasmid pDAW200 (generously provided by Professor Dr Ari Helenius and Dr Bruno Martoglio, ETH Zurich, Switzerland) was used to generate the hspp gene. hspp was amplified by PCR with primers 5'-CTTGTTGAATTCCGAGCTCACTAGTTCACC-3' and 5'-AGAACTCTAGAGCTTCCTGAGAGCTCGGC-3'. DNA products were then digested by EcoRI and XbaI and ligated into pPIC6 B (Invitrogen) to create the clone hspp. The vector containing the hspp gene was linearized by digestion with PmeI. The DNA clone was introduced by electroporation into P. pastoris that already contained the C or CE1E2 gene and transformants were selected on blasticidin plates (Invitrogen). Recombinant proteins were induced with 1 % methanol and extracted as described previously (Majeau et al., 2004).
SDS-PAGE and Western blotting.
After lysis, protein extracts were denatured by mixing with an equal amount of buffer-saturated phenol and vortexing for 2 min. The hydrophobic phase was recovered after centrifugation at 20 000 g for 5 min and mixed with 5 vols methanol/0·1 M ammonium acetate solution. Proteins were recovered after 1 h incubation at 20 °C and spun at 13 000 r.p.m. for 5 min. Pellets were washed with methanol and, after drying, mixed with SDS loading dye. Tris/glycine and Tris/Bicine SDS-PAGE were performed as described by Lemberg & Martoglio (2003). After migration, proteins were transferred to nitrocellulose membranes by using a Trans-Blot apparatus (Bio-Rad) and revealed with an anti-C antibody (C1170; Majeau et al., 2004
) or with anti-hspp antibodies (generously provided by Dr Todd E. Golde, Mayo Clinic, Jacksonville, FL, USA).
Density-gradient centrifugation.
After lysis, yeast extracts were spun at 20 000 g and the supernatant was passed through a 0·2 µm filter. Protein concentration was determined by BCA protein assays (Pierce) and equal loads of protein were layered onto a continuous (1060 % w/w) sucrose gradient [50 mM sodium citrate, pH 6, and 300 mM NaCl (final concentration)] and centrifuged for 20 h at 4 °C at 120 000 g in an NVT65 rotor (Beckman). For some experiments, similar amounts of C as determined by ELISA were separated by ultracentrifugation. Fractions were collected, mixed with NP-40 (0·6 %) and analysed by ELISA using a polyclonal rabbit antibody against HCV C protein (C1170) (Majeau et al., 2004). When stated, yeast extracts were incubated with 0·6 % NP-40 for 30 min at 4 °C before loading onto the sucrose gradient. ER membranes were labelled by inducing yeast in medium supplemented with 0·1 µM ER-Tracker Green (glibenclamide BODIPY FL; Invitrogen). Fluorescence in the sucrose fraction was analysed on a Typhoon 9200 fluorescence imager (Amersham Biosciences). To measure intact mitochondria in the gradient fractions, 2·5 µM MitoTracker Red CM-H2XRos (Invitrogen) was mixed with 100 µl sample diluted in 200 µl PBS and incubated at 25 °C for 1 h. Fluorescence was detected on a Typhoon 9200 imager (Amersham Biosciences).
Electron microscopy (EM).
Samples were absorbed onto 400-mesh carbonFormvar grids (Canemco) for 5 min. Grids were washed with TBS and stained for 5 min with filtered 2 % (w/v) uranyl acetate. Immunogold labelling was performed as described previously (Majeau et al., 2004), using anti-hspp antibodies. Grids were examined under an electronic microscope with an acceleration voltage of 60 kV at a magnification of x100 000.
Immunocapture.
After sucrose-gradient centrifugation, protein samples (100 µl) were diluted in PBS (pH 7·2) and mixed with magnetic beads (Dynabead m-280) conjugated to a sheep anti-rabbit antibody that had previously been incubated for 30 min with rabbit anti-hspp antibody, and then washed carefully. After 1 h incubation at 4 °C, protein mixes were exposed to magnetic forces and washed three times with PBS. SDS loading dye was added to the tube and extracted proteins were analysed by Western blot using anti-C antibody as described above.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
hspp activity was revealed in a time-course experiment where C and hspp were expressed together with a methanol-inducible promoter (Fig. 2). A single band of 23 kDa was observed when the C construct (1191) was expressed alone (Fig. 2a
). The protein remained unprocessed even 72 h after induction (data not shown). However, co-expression of C (1191) with hspp led to the appearance of a processed form (21 kDa) 1 h after induction (Fig. 2b
). The 23 kDa precursor had been processed completely to 21 kDa, 12 h after induction of both proteins. In the context of the CE1E2 polyprotein, we observed that C was processed to the 21 kDa form by the yeast-encoded spp enzyme. However, co-expression of hspp accelerated the maturation of C; the maturation was completed after 3 h co-expression (Fig. 2d
), instead of after 24 h when CE1E2 was expressed alone (Fig. 2c
).
|
|
It has previously been described that, after cleavage by spp, the C protein associates with the outer membranes of the mitochondria (Schwer et al., 2004). In order to detect these organelles, we mixed MitoTracker Red CM-H2XRos, a reduced dye that becomes fluorescent when entering an actively respiring mitochondrion, with the sucrose-gradient fractions. We detected fluorescence in the fractions with densities of 1·161·20 g ml1, fractions previously described to contain mitochondria (Lee et al., 1969
; Walworth et al., 1989
) and corresponding to the second peak of mature C protein. As for ER membranes, they were detected in the sucrose fraction by using glibenclamide conjugated to a fluorochrome (ER-Tracker Green dye). Glibenclamide binds to the sulphonylurea receptors of ATP-sensitive K+ channels, which are prominent on the ER. This dye is highly selective for the ER and rarely stains mitochondria. Yeast expressing the C construct (1191), hspp or both was induced in medium containing 0·1 µM ER-Tracker Green dye for 24 h. Extracts were separated on a sucrose gradient and fluorescence in the samples was evaluated on a phosphorimager. As shown in Fig. 3(b)
, in the hspp extract, ER membranes were present at densities of 1·161·20 g ml1, as for the mitochondria. However, the presence of C shifted some of the ER membranes to less-dense fractions. ER membranes are expected to be associated with the NLPs with a density of 1·11 g ml1.
Particles of different densities in the fractions were visualized by EM (Fig. 4). In fractions with a density of 1·11 g ml1 from C (1191)-expressing cells, particles of 3560 nm in diameter were observed (Fig. 4a
), suggesting that the immature protein of 23 kDa can assemble into NLPs. These particles were similar to C (1191)/hspp NLPs present at the same density (Fig. 4b
) and resembled enveloped virus particles isolated from infected patients (Kaito et al., 1994
). Non-enveloped NLPs from C (1191)/hspp-expressing cells, detected at a density of 1·25 g ml1, showed a diameter of 2845 nm (Fig. 4c
), i.e. within the size range of non-enveloped particles characterized previously (Maillard et al., 2001
). In the 1·17 g ml1 density fraction, we observed membranes, aggregated material and mitochondria, but NLPs were not found.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The resistance of C (1191) to cleavage by the yeast spp allowed us to show that the maturation of C is not essential for the budding process and that immature C protein is able to recruit membrane around the NLPs. We observed that immature full-length C can generate NLPs of 3560 nm diameter with a density of 1·11 g ml1. Treatment with NP-40 removed the envelope and shifted the NLPs to a density of 1·25 g ml1, as shown for HCV particles isolated from the serum of infected patients (Miyamoto et al., 1992; Kanto et al., 1994
; Ishida et al., 2001
). However, the C protein detected in the blood of infected patients was present only as the mature 21 kDa form (Yasui et al., 1998
). Interestingly, we did not find free C protein associated with the rough ER membrane fraction occurring at a density of 1·18 g ml1 (Walworth et al., 1989
), suggesting that encapsidation and NLP formation are processes that occur rapidly after synthesis.
We observed that the yield of C in yeast was higher when the protein was co-expressed with hspp. It is likely that overexpression of C alone leads to clogging in the ER, which affects the fitness of the yeast and thus the yield of protein. The maturation of C with hspp promotes the release of C from this compartment, probably releasing the physiological stress on the cell.
Co-expression of hspp in yeast led to efficient processing of C into its mature form. The processed C protein was found at three different densities: 1·11 g ml1 (enveloped NLPs), 1·17 g ml1 (free C protein) and 1·25 g ml1 (non-enveloped NLPs). It has previously been proposed that C, after hspp cleavage, travels along the ER membrane and reaches the surface of lipid droplets that are formed between the two layers of the ER membrane (McLauchlan et al., 2002) or diffuses to the surface of MAM (Schwer et al., 2004
). Yeast produces a small number of cytosolic lipid bodies (Murphy & Vance, 1999
). This shortage in lipid droplets may favour accumulation of C on the surface of mitochondrial membranes. The MAM fraction is described as fractionating at a density of 1·171·20 g ml1 after sucrose-gradient centrifugation (Rusiñol et al., 1994
). The presence of C protein in the 1·17 g ml1 fraction probably reflects association with these structures.
Non-enveloped particles have been detected in blood and in hepatocytes (Miyamoto et al., 1992). The degree of liver inflammation also influences the number of non-enveloped virions circulating in the blood (Kanto et al., 1994
). Here, we demonstrate that maturation of the C protein by hspp is essential for the production of free nucleocapsid in the cells. C protein was only present in the 1·25 g ml1 fraction when hspp was co-expressed. The accumulation of NLPs also correlated with the amount of hspp expressed in yeast. These results suggest that, after maturation by hspp, C protein is free to move from the ER membranes and becomes organized in NLPs without any lipid envelope. This capacity to self-assemble without ER membranes has been observed in in vitro experiments where the purified protein mixed with structured RNA was sufficient for producing particles (Kunkel et al., 2001
; Lorenzo et al., 2001
; Majeau et al., 2004
).
Protein concentration is important in the initiation of NLP formation. C needs to be expressed at a very high level to trigger the formation of NLPs in cellular systems (Baumert et al., 1998; Blanchard et al., 2002
; Ezelle et al., 2002
; Greive et al., 2002
). As the C protein is first trapped in enveloped NLPs, only a small amount of mature protein is available for the formation of non-enveloped NLPs. This can account for the late production of non-enveloped NLPs after expression.
Several viruses carry the proteases necessary for maturation of their nucleocapsid within the virus particle itself (Greber et al., 1996; Andrés et al., 2001
). Similarly, our experiments show that hspp is sequestered within the membrane of enveloped NLPs. In sedimentation experiments, we observed a shift of hspp toward the fraction of enveloped particles. C was also pulled down when samples were incubated with anti-spp antibody. It was shown previously that hspp is not associated directly with the C protein (Okamoto et al., 2004
). We propose that immature C protein triggers the formation of a virion-like structure, incorporating ER membrane via its C-terminal anchoring domain, thus capturing hspp associated with the ER (Fig. 8
). The capsid possibly comprises both mature and immature protein. Completion of the maturation of the virus particle can then occur after the budding process.
|
The amount of hspp protein is probably an important factor in the infection process. It is likely that a large proportion of hspp is sequestered in budding virions, simultaneously reducing the amount of the enzyme in the ER compartment of the infected cells. The lack of hspp can be toxic for the cells (Wu & Chang, 2004) and can impair the immune response by affecting the maturation and migration to the surface of major histocompatibility complex (MHC) class I antigens (Bland et al., 2003
). Consistent with this observation, it was shown recently that MHC class I presentation by dendritic cells was impaired in transgenic mice expressing HCV structural proteins (Hiasa et al., 2004
).
In summary, we have shown that the level of hspp protein influences the number of non-enveloped particles. The major HCV populations are described as changing from virions to nucleocapsids with the progression of liver disease or inflammation (Kanto et al., 1995; Watson et al., 1996
). It is possible that a change in hspp expression occurs during HCV infection and that this contributes to an increase in non-enveloped particles in infected cells. We are currently investigating the effect of HCV infection on hspp in infected patients.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acosta-Rivero, N., Musacchio, A., Lorenzo, L., Alvarez, C. & Morales, J. (2002). Processing of the hepatitis C virus precursor protein expressed in the methylotrophic yeast Pichia pastoris. Biochem Biophys Res Commun 295, 8184.[CrossRef][Medline]
Acosta-Rivero, N., Falcón, V., Alvarez, C. & 12 other authors (2003). Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast. Biochem Biophys Res Commun 310, 4853.[CrossRef][Medline]
Alejo, A., Andrés, G. & Salas, M. L. (2003). African swine fever virus proteinase is essential for core maturation and infectivity. J Virol 77, 55715577.
André, P., Komurian-Pradel, F., Deforges, S. & 7 other authors (2002). Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol 76, 69196928.
Andrés, G., Alejo, A., Simón-Mateo, C. & Salas, M. L. (2001). African swine fever virus protease, a new viral member of the SUMO-1-specific protease family. J Biol Chem 276, 780787.
Barba, G., Harper, F., Harada, T. & 8 other authors (1997). Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc Natl Acad Sci U S A 94, 12001205.
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.
Bland, F. A., Lemberg, M. K., McMichael, A. J., Martoglio, B. & Braud, V. M. (2003). Requirement of the proteasome for the trimming of signal peptide-derived epitopes presented by the nonclassical major histocompatibility complex class I molecule HLA-E. J Biol Chem 278, 3374733752.
Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. M. & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359362.[Medline]
Choo, S.-H., So, H.-S., Cho, J. M. & Ryu, W.-S. (1995). Association of hepatitis C virus particles with immunoglobulin: a mechanism for persistent infection. J Gen Virol 76, 23372341.[Abstract]
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]
Ezelle, H. J., Markovic, D. & Barber, G. N. (2002). Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector. J Virol 76, 1232512334.
Falcón, V., García, C., de la Rosa, M. C., Menéndez, I., Seoane, J. & Grillo, J. M. (1999). Ultrastructural and immunocytochemical evidences of core-particle formation in the methylotrophic Pichia pastoris yeast when expressing HCV structural proteins (core-E1). Tissue Cell 31, 117125.[CrossRef][Medline]
Falcón, V., Acosta-Rivero, N., Chinea, G. & 12 other authors (2003). Ultrastructural evidences of HCV infection in hepatocytes of chronically HCV-infected patients. Biochem Biophys Res Commun 305, 10851090.[CrossRef][Medline]
Golde, T. E. & Younkin, S. G. (2001). Presenilins as therapeutic targets for the treatment of Alzheimer's disease. Trends Mol Med 7, 264269.[CrossRef][Medline]
Greber, U. F., Webster, P., Weber, J. & Helenius, A. (1996). The role of the adenovirus protease in virus entry into cells. EMBO J 15, 17661777.[Medline]
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]
Hiasa, Y., Takahashi, H., Shimizu, M., Nuriya, H., Tsukiyama-Kohara, K., Tanaka, T., Horiike, N., Onji, M. & Kohara, M. (2004). Major histocompatibility complex class-I presentation impaired in transgenic mice expressing hepatitis C virus structural proteins during dendritic cell maturation. J Med Virol 74, 253261.[CrossRef][Medline]
Hijikata, M., Shimizu, Y., Kato, H., Iwamoto, A., Shih, J. W., Alter, H. J., Purcell, R. H. & Yoshikura, H. (1993). Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes. J Virol 67, 19531958.[Medline]
Hüssy, P., Langen, H., Mous, J. & Jacobsen, H. (1996). Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology 224, 93104.[CrossRef][Medline]
Ishida, S., Kaito, M., Kohara, M., Tsukiyama-Kohora, K., Fujita, N., Ikoma, J., Adachi, Y. & Watanabe, S. (2001). Hepatitis C virus core particle detected by immunoelectron microscopy and optical rotation technique. Hepatol Res 20, 335347.[CrossRef][Medline]
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]
Kanto, T., Hayashi, N., Takehara, T., Hagiwara, H., Mita, E., Naito, M., Kasahara, A., Fusamoto, H. & Kamada, T. (1994). Buoyant density of hepatitis C virus recovered from infected hosts: two different features in sucrose equilibrium density-gradient centrifugation related to degree of liver inflammation. Hepatology 19, 296302.[CrossRef][Medline]
Kanto, T., Hayashi, N., Takehara, T., Hagiwara, H., Mita, E., Naito, M., Kasahara, A., Fusamoto, H. & Kamada, T. (1995). Density analysis of hepatitis C virus particle population in the circulation of infected hosts: implications for virus neutralization or persistence. J Hepatol 22, 440448.[CrossRef][Medline]
Kato, T., Miyamoto, M., Furusaka, A., Date, T., Yasui, K., Kato, J., Matsushima, S., Komatsu, T. & Wakita, T. (2003). Processing of hepatitis C virus core protein is regulated by its C-terminal sequence. J Med Virol 69, 357366.[CrossRef][Medline]
Kiernan, R. E., Ono, A., Englund, G. & Freed, E. O. (1998). Role of matrix in an early postentry step in the human immunodeficiency virus type 1 life cycle. J Virol 72, 41164126.
Kunkel, M., Lorinczi, M., Rijnbrand, R., Lemon, S. M. & Watowich, S. J. (2001). Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein. J Virol 75, 21192129.
Lauer, G. M. & Walker, B. D. (2001). Hepatitis C virus infection. N Engl J Med 345, 4152.
Lee, T. C., Swartzendruber, D. C. & Snyder, F. (1969). Zonal centrifugation of microsomes from rat liver: resolution of rough- and smooth-surfaced membranes. Biochem Biophys Res Commun 36, 748755.[CrossRef][Medline]
Lemberg, M. K. & Martoglio, B. (2003). Analysis of polypeptides by sodium dodecyl sulfatepolyacrylamide gel electrophoresis alongside in vitro-generated reference peptides. Anal Biochem 319, 327331.[CrossRef][Medline]
Lin, C., Lindenbach, B. D., Prágai, B. M., McCourt, D. W. & Rice, C. M. (1994). Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol 68, 50635073.[Medline]
Lorenzo, L. J., Dueñas-Carrera, S., Falcón, V., Acosta-Rivero, N., González, E., de la Rosa, M. C., Menéndez, I. & Morales, J. (2001). Assembly of truncated HCV core antigen into virus-like particles in Escherichia coli. Biochem Biophys Res Commun 281, 962965.[CrossRef][Medline]
Maillard, P., Krawczynski, K., Nitkiewicz, J. & 7 other authors (2001). Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients. J Virol 75, 82408250.
Majeau, N., Gagné, V., Boivin, A., Bolduc, M., Majeau, J.-A., Ouellet, D. & Leclerc, D. (2004). The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation. J Gen Virol 85, 971981.
Martoglio, B. & Golde, T. E. (2003). Intramembrane-cleaving aspartic proteases and disease: presenilins, signal peptide peptidase and their homologs. Hum Mol Genet 12, R201R206.
McLauchlan, J., Lemberg, M. K., Hope, G. & Martoglio, B. (2002). Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J 21, 39803988.[CrossRef][Medline]
Miyamoto, H., Okamoto, H., Sato, K., Tanaka, T. & Mishiro, S. (1992). Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction. J Gen Virol 73, 715718.[Abstract]
Moradpour, D., Brass, V., Gosert, R., Wölk, B. & Blum, H. E. (2002). Hepatitis C: molecular virology and antiviral targets. Trends Mol Med 8, 476482.[CrossRef][Medline]
Murphy, D. J. & Vance, J. (1999). Mechanisms of lipid-body formation. Trends Biochem Sci 24, 109115.[CrossRef][Medline]
Nyborg, A. C., Kornilova, A. Y., Jansen, K., Ladd, T. B., Wolfe, M. S. & Golde, T. E. (2004). Signal peptide peptidase forms a homodimer that is labeled by an active site-directed -secretase inhibitor. J Biol Chem 279, 1515315160.
Ogino, T., Fukuda, H., Imajoh-Ohmi, S., Kohara, M. & Nomoto, A. (2004). Membrane binding properties and terminal residues of the mature hepatitis C virus capsid protein in insect cells. J Virol 78, 1176611777.
Okamoto, K., Moriishi, K., Miyamura, T. & Matsuura, Y. (2004). Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein. J Virol 78, 63706380.
Prince, A. M., Huima-Byron, T., Parker, T. S. & Levine, D. M. (1996). Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins. J Viral Hepat 3, 1117.[Medline]
Roingeard, P., Hourioux, C., Blanchard, E., Brand, D. & Ait-Goughoulte, M. (2004). Hepatitis C virus ultrastructure and morphogenesis. Biol Cell 96, 103108.[CrossRef][Medline]
Rusiñol, A. E., Cui, Z., Chen, M. H. & Vance, J. E. (1994). A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre-Golgi secretory proteins including nascent lipoproteins. J Biol Chem 269, 2749427502.
Sakuragi, S., Goto, T., Sano, K. & Morikawa, Y. (2002). HIV type 1 Gag virus-like particle budding from spheroplasts of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 99, 79567961.
Santolini, E., Migliaccio, G. & La Monica, N. (1994). Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol 68, 36313641.[Medline]
Schwartz, M., Chen, J., Lee, W.-M., Janda, M. & Ahlquist, P. (2004). Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication. Proc Natl Acad Sci U S A 101, 1126311268.
Schwer, B., Ren, S., Pietschmann, T., Kartenbeck, J., Kaehlcke, K., Bartenschlager, R., Yen, T. S. B. & Ott, M. (2004). Targeting of hepatitis C virus core protein to mitochondria through a novel C-terminal localization motif. J Virol 78, 79587968.
Suzuki, R., Tamura, K., Li, J., Ishii, K., Matsuura, Y., Miyamura, T. & Suzuki, T. (2001). Ubiquitin-mediated degradation of hepatitis C virus core protein is regulated by processing at its carboxyl terminus. Virology 280, 301309.[CrossRef][Medline]
Thomssen, R. & Bonk, S. (2002). Virolytic action of lipoprotein lipase on hepatitis C virus in human sera. Med Microbiol Immunol (Berl) 191, 1724.[CrossRef][Medline]
Walworth, N. C., Goud, B., Ruohola, H. & Novick, P. J. (1989). Fractionation of yeast organelles. Methods Cell Biol 31, 335356.[Medline]
Watson, J. P., Bevitt, D. J., Spickett, G. P., Toms, G. L. & Bassendine, M. F. (1996). Hepatitis C virus density heterogeneity and viral titre in acute and chronic infection: a comparison of immunodeficient and immunocompetent patients. J Hepatol 25, 599607.[CrossRef][Medline]
Weihofen, A., Binns, K., Lemberg, M. K., Ashman, K. & Martoglio, B. (2002). Identification of signal peptide peptidase, a presenilin-type aspartic protease. Science 296, 22152218.
Wu, C.-M. & Chang, M. D.-T. (2004). Signal peptide of eosinophil cationic protein is toxic to cells lacking signal peptide peptidase. Biochem Biophys Res Commun 322, 585592.[CrossRef][Medline]
Yasui, K., Wakita, T., Tsukiyama-Kohara, K., Funahashi, S.-I., Ichikawa, M., Kajita, T., Moradpour, D., Wands, J. R. & Kohara, M. (1998). The native form and maturation process of hepatitis C virus core protein. J Virol 72, 60486055.
Received 10 May 2005;
accepted 19 August 2005.