MRC Virology Unit, Church Street, Glasgow G11 5JR, UK
Correspondence
Arvind H. Patel
a.patel{at}vir.gla.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The C terminus of each structural protein is composed of a hydrophobic amino acid sequence, which acts as a signal peptide to target the proteins located downstream to the endoplasmic reticulum (ER). Cleavage at the C/E1, E1/E2, E2/p7 and p7/NS2 sites is mediated by ER-resident host signal peptidase(s) (Lindenbach & Rice, 2001; Rosenberg, 2001
). Although cleavage at the C/E1 and E1/E2 sites proceeds to completion rapidly after translation, cleavage at E2/p7 and p7/NS2 is delayed, resulting in an E2p7NS2 species (Dubuisson et al., 1994
). Furthermore, cleavage at the E2/p7 site has been shown to be incomplete, resulting in two E2-specific species, E2 and E2p7 (Lin et al., 1994
; Mizushima et al., 1994
). The significance of these two forms of E2 has not been established, although it is conceivable that both play functional roles in the virus life cycle.
The inability to culture HCV remains a major obstacle to the study of virus assembly. HCV virus-like particle (VLP) assembly can occur in the absence of p7 upon expression of C, E1 and E2 in insect (but not mammalian) cells (Baumert et al., 1998, 1999
; Clayton et al., 2002
; Owsianka et al., 2001
). A homologue of p7 (but not the E2p7 species) in the related pestivirus, bovine viral diarrhea virus (BVDV), is required for the production of infectious virus progeny (Harada et al., 2000
). By analogy with BVDV p7 and its presumed role as an ion channel, it is possible that HCV p7 is important for virus particle morphogenesis.
HCV p7 is an integral membrane protein, which is translocated into the ER by the signal peptide located in the C terminus of E2 (Carrere-Kremer et al., 2002; Cocquerel et al., 2002
). When expressed on its own in mammalian cells, p7 has been shown to have two membrane-spanning domains with its N and C termini luminally disposed and a short hydrophilic loop facing the cytosol. The second transmembrane (TM) domain of p7 can act as a signal peptide to target NS2 to the ER, although the membrane association of NS2 can occur independently of any p7 sequences (Yamaga & Ou, 2002
).
In this study, we investigated the proteolytic processing and TM topology of p7 when expressed in the context of E2p7. We have shown that the C terminus of the E2p7 species is cytoplasmically orientated, indicating that p7 adopts a dual TM topology and that processing at the E2/p7 site is influenced by sequences within the signal peptide of p7 and also sequences in the NS2 region.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Transfection of cultured cells.
Huh-7 cells were infected with vTF7-3, an rVV expressing T7 RNA polymerase (Fuerst et al., 1986), at an m.o.i. of 5 for 1 h at 37 °C. The cells were then transfected with appropriate plasmids using a liposome-mediated method (Rose et al., 1991
). Following incubation for 18 h at 37 °C, cells were washed with PBS, lysed in lysis buffer (20 mM Tris/HCl pH 7·4, 20 mM iodoacetamide, 150 mM NaCl, 1 mM EDTA, 0·5 % Triton X-100) and the lysate spun briefly to remove nuclei. The clarified lysates were subjected to Western immunoblotting using appropriate antibodies and bound antibodies were detected using enhanced chemiluminescence reagents (Amersham). The quantification of protein bands in the Western immunoblot was performed using Quantity One Volume Analysis software (Bio-Rad).
Radiolabelling of proteins.
Huh-7 cells were infected with appropriate rVVs at an m.o.i. of 10 and incubated for 6 h at 37 °C. Infected cells were washed with PBS, incubated in methionine-free medium containing 50 µCi (1·85 MBq) [35S]methionine ml1 for 18 h and the cell lysate prepared as described above. To immunoprecipitate proteins, radiolabelled cell lysates were incubated with anti-E2 or anti-c-myc antibodies for 2 h at 4 °C and the immune complexes precipitated by incubation at 4 °C for 2 h with protein ASepharose equilibrated with lysis buffer. Immune complexes attached to protein ASepharose were washed five times with lysis buffer and bound proteins were either directly released into SDS-PAGE denaturing buffer or were subjected to peptide N-glycosidase F (PNGase F) (New England Biolabs) treatment following the manufacturer's protocol. Immune complexes were subjected to 10 % SDS-PAGE. Gels were dried and exposed overnight to a phosphor screen and radiolabelled proteins were visualized with a Bio-Rad Personal FX phosphorimager.
Antibodies.
Hybridomas secreting the mouse monoclonal antibody (mAb) 9E10 (Evan et al., 1985) to human c-myc product were obtained from the European Collection of Cell Cultures. The anti-E2 antibodies AP33 and ALP98, and the rabbit polyclonal antiserum R646 have been described previously (Clayton et al., 2002
; Owsianka et al., 2001
). The anti-E2 mAb H53 was kindly supplied by J. Dubuisson (Cocquerel et al., 1998
).
VLP preparation.
VLPs from Sf21 cells infected with rbacs were prepared essentially as described by Baumert et al. (1998). The purity and quality of the VLP preparation were verified by negative-stained transmission electron microscopy (EM) as follows. VLPs (5 µl) were loaded on to Formvar-coated nickel grids, stained with Nanovan (Nanoprobe) and examined under a JEOL 100 S electron microscope. For immunogold labelling, samples loaded on to Formvar-coated nickel grids were incubated in primary antibody for 23 h at room temperature. The grids were washed three times in distilled water and incubated for 2 h with anti-mouse IgG conjugated to 10 nm gold particles (Nanoprobe). Following three washes as above, samples were stained with Nanovan and examined by EM.
Trypsin protection analysis.
Huh-7 cells were infected with appropriate rVVs and proteins radiolabelled as described above. Following incubation, cells were rinsed once with ice-cold PBS, twice with homogenization buffer (10 mM HEPES pH 7·4, 1 mM EDTA, 1 mM PMSF, 0·25 M sucrose) and lysed in this buffer using a tight-fitting Dounce homogenizer. After a brief centrifugation, the post-nuclear supernatant containing microsomal membranes was overlaid on a cushion of 0·6 M sucrose in 10 mM HEPES pH 7·4, 1 mM EDTA, 1 mM PMSF and centrifuged at 20 000 r.p.m. in a Sorval TH641 rotor. The pellet containing microsomal membrane was washed and resuspended in PBS. For trypsin protection analysis, the microsomal preparation was divided into three equal aliquots. One sample was left untreated while the remaining two were treated with 50 µg freshly prepared trypsin (Sigma) ml1 in the presence or absence of 1 % NP-40 for 1 h at 37 °C. Proteolysis was halted by the addition of 30 µg aprotinin (Sigma) ml1 and further incubation on ice for 1 h. NP-40 was added to all samples to a final concentration of 1 % and incubated on ice for 1 h. Samples were spun at 13 000 r.p.m. for 5 min, the resultant supernatant subjected to immunoprecipitation using mAb ALP98 or 9E10 and the immune complexes analysed by SDS-PAGE.
Immunofluorescence assay.
Huh-7 cells grown on coverslips were transfected as described above (but in the absence of vTF7.3 infection) with the plasmid expressing E1E2p7c-myc. Following incubation at 37 °C for 48 h, cells were washed with PBS, fixed with 2 % paraformaldehyde for 5 min at room temperature and permeabilized, either selectively or completely, as described by Crystal et al. (2003). Briefly, selective permeabilization was performed by incubating cells with 200 U streptolysin O (SLO; Sigma) ml1 for 5 min at room temperature followed by rinsing with PBS. For complete solubilization, cells were incubated with 0·1 % NP-40 at room temperature for 10 min and then washed with PBS. All cells were blocked with PBS containing 2 % FCS for 20 min at room temperature and then probed with the anti-E2 rabbit antiserum R646 and anti-c-myc mAb 9E10 for 2 h at room temperature. Following three washes with PBS, cells were incubated at room temperature for 2 h with Alexa Fluor 633 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) as appropriate. Following final washes with PBS, coverslips were mounted on glass slides and examined under a Zeiss laser-scanning confocal microscope. The fluorescent images were analysed using the LSM510 software.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Radiolabelled proteins from Huh-7 cells infected with rVVs expressing various HCV sequences (Fig. 1) were immunoprecipitated with the anti-E2 mAb ALP98 or the anti-c-myc mAb 9E10 and analysed by SDS-PAGE, either directly or following treatment with PNGase F, a glycosidase that removes all N-linked sugar moieties attached to glycoproteins. As shown in Fig. 2
(a, lanes 16), the anti-E2 mAb immunoprecipitated E2 species of different molecular masses from cells infected with various rVVs, indicating differences in glycosylation and/or processing at the E2/p7 site. The differences in the relative SDS-PAGE mobilities of the E2 species synthesized in cells infected with various rVVs were better resolved when the immunoprecipitated proteins were treated with PNGase F (Fig. 2a
, lanes 916). Thus, rVVs expressing the full-length (FL) ORF and E1E2p7NS2t (Fig. 2a
, lanes 10 and 11) produced both a fully processed E2 that co-migrated with the product synthesized by rVV-E1E2 (Fig. 2a
, lane 12) and a slower-migrating form representing E2p7, indicating partial processing at the E2/p7 site. Cells infected with rVV-E1E2p7 and rVV-E1E2p7c-myc produced predominantly E2p7 and E2p7 plus E2p7c-myc, respectively, as well as a small amount of fully processed E2 (Fig. 2a
, lanes 13 and 14). The relative amount of fully processed E2 was higher in cells expressing the FL ORF or E1E2p7NS2t compared with those lacking sequences downstream from p7 (Fig. 2a
, lanes 1014). Taken together, these results are in keeping with previously published observations that proteolytic processing at the E2/p7 site is incomplete in the genotype 1a sequence (Lin et al., 1994
; Mizushima et al., 1994
) and also indicated that sequences downstream of p7 may enhance cleavage efficiency at this site.
|
To test whether E2p7 or E2p7c-myc formed a heterodimer complex with E1, radiolabelled extracts of cells expressing various HCV sequences were immunoprecipitated with the conformation-sensitive anti-E2 mAb H53 (which specifically recognizes the native E1E2 complex but not the disulfide-linked aggregate) (Cocquerel et al., 1998; Duvet et al., 1998
) or the anti-c-myc mAb 9E10. Analysis of the corresponding immune complexes confirmed that both E2p7 and E2p7c-myc formed a heterodimer with E1 (Fig. 2b
, lanes 5, 6 and 8). As expected, the native E1E2 heterodimer was also detected in cells expressing E1E2, E2E2p7NS2t and the FL ORF (Fig. 2b
, lanes 24). That E2p7c-myc interacted with E1 was further confirmed using an anti-E1 antiserum, which specifically co-immunoprecipitated this species (data not shown).
Processing at the E2/p7 site is deficient in an insect cell system
Previous studies have shown assembly of HCV VLPs upon expression of viral structural proteins (C, E1 and E2) in insect cells infected with rbac (Baumert et al., 1998; Owsianka et al., 2001
). Here, as part of our interest in understanding the mechanisms of VLP assembly, we compared the characteristics of HCV structural proteins expressed in insect cells as CE1E2, CE1E2p7 or CE1E2p7c-myc. Radiolabelled proteins from Sf21 cells infected with different rbacs were immunoprecipitated with an anti-E2 antiserum and the immune complexes treated with PNGase F. As shown in Fig. 3(a)
, E2 species representing E2, E2p7 or E2p7c-myc from cells infected with rbacs encoding CE1E2, CE1E2p7 or CE1E2p7c-myc, respectively, were immunoprecipitated (Fig. 3a
, lanes 24). Interestingly, no processed E2 was observed in cells infected with rbac-CE1E2p7 or rbac-CE1E2p7c-myc, and a substantial portion of c-myc remained a part of E2p7c-myc. As seen in Huh-7 cells (Fig. 2a
, lane 16), there was processing at the p7/c-myc junction, resulting in E2p7c-myc and E2p7 products (Fig. 3a
, lane 4).
|
Mutational analysis of the sequence that acts as a signal peptide for p7
It has previously been reported that processing at the E2/p7 site of the genotype 1b strain BK is more efficient than that in genotype 1a strain H (Lin et al., 1994), an isolate closely related to strain H77c used in this study. The C terminus (aa 731746) of E2 acts as a signal peptide to translocate p7 into the ER (Carrere-Kremer et al., 2002
; Cocquerel et al., 2002
). Presumably specific amino acid residues present in this region also play a role in cleavage between E2 and p7. Comparison of the signal peptide sequence present at the C terminus of E2 of strains BK (Takamizawa et al., 1991
) and H77c (Yanagi et al., 1997
) revealed 3 aa differences (Fig. 4a
). To test whether the changes L720V, A733S and A742S in the H77c sequence relative to that in the BK strain were responsible for the genotype-dependent differences in the efficiency of processing at the E2/p7 site, we substituted valine, serine and serine at residues 720, 733 and 742 for leucine (V720L), alanine (S733A) and alanine (S742A), respectively. In addition, another substitution mutation, valine to isoleucine at position 719 (V719I), was introduced inadvertently into some of the constructs. These mutations were introduced individually and in different combinations, into the construct expressing the strain H77c-encoded E1E2p7NS2t. Plasmids encoding E1E2, E1E2p7NS2t or those carrying different substitutions were transfected in rVV vTF7.3-infected Huh-7 cells. Eighteen hours after transfection, cell lysates were subjected to PNGase F treatment followed by Western immunoblotting using an anti-E2 antiserum. As seen previously in Fig. 2(a)
, cells expressing unmodified E1E2p7NS2t synthesized E2p7 and E2, with the latter species co-migrating with E2 expressed in cells transfected with the construct encoding E1E2 (Fig. 4b
). The level of processed E2 was 55 % of the total amount of the two products. A similar E2p7 : E2 ratio was found with the substitution V719I, whereas slightly reduced amounts of processed E2 were seen with S733A+S742A and S742A mutations. In contrast, mutations involving aa 720, either alone (V720L) or together with the other residue substitutions (V719I+V720L, V719I+V720L+S733A, V719I+V720L+S733A+S742A), produced significantly increased amounts of E2 species relative to E2p7. This indicated that leucine at position 720 plays a crucial role in proteolytic processing at the E2/p7 site. In addition, isoleucine at position 719 and the two alanines at positions 733 and 742 acted co-operatively with L720 to enhance processivity at the E2/p7 site further.
|
To investigate the TM topology of inefficiently processed p7, trypsin protection assays of radiolabelled microsomes prepared from Huh-7 cells infected with rVV-E1E2p7 or rVV-E1E2p7c-myc were performed. Microsomes were either untreated or treated with trypsin in the presence or absence of 1 % NP-40 prior to immunoprecipitation with anti-E2 or anti-c-myc mAbs and analysed. As shown in Fig. 5(a, lanes 1, 2, 4 and 5), mAb ALP98 recognized E2 (and co-precipitated E1) in untreated or trypsin-treated microsomes of cells infected with both rVVs. Interestingly, the E2 band precipitated by mAb ALP98 from the microsomes of rVV-E1E2p7c-myc-infected cells treated with trypsin in the absence of detergent migrated at a slightly faster rate than that from the corresponding untreated sample (Fig. 5a
, lanes 5 and 4, respectively) (the difference in the molecular mass was more apparent at low exposure). This indicated possible trimming of E2p7c-myc, most probably at the C-terminal end. As expected, neither E1 nor E2 was observed in the trypsin-treated detergent-solubilized microsomes (Fig. 5a
, lanes 3 and 6). The epitope recognized by mAb ALP98 is located within the E2 ectodomain (Clayton et al., 2002
), which is luminally disposed, and therefore is expected to be available for trypsin degradation only in detergent-solubilized microsomes. Our mAb ALP98 results shown in Fig. 5(a)
are in accordance with this hypothesis. In contrast to the mAb ALP98 data, immunoprecipitation with the anti-c-myc mAb showed that the E2p7 species produced by rVV-E1E2p7c-myc was degraded upon treatment of microsomes in the absence as well as in the presence of detergent (Fig. 5a
, lanes 8 and 9), indicating the c-myc epitope tag to be cytoplasmically exposed. Interestingly, a trypsin-resistant fragment of approximately 18 kDa was seen following treatment of microsomes from cells expressing E1E2p7c-myc with trypsin in the absence of detergent (Fig. 5a
, lane 8).
|
A small central portion (aa 779781) of p7 has been shown to be cytoplasmically orientated (Carrere-Kremer et al., 2002). Therefore, it is possible that trypsin in the biochemical assays above may have cleaved at this cytoplasmic loop, thus generating a fragment too small to be observed on the gel, hence allowing the possibility of the c-myc tag being disposed on the luminal side of the membrane. To rule out this possibility, immunofluorescence microscopy was performed under different permeabilization conditions. Huh-7 cells infected with rVV-E1E2p7c-myc were fixed and treated with 1 % NP-40 to permeabilize all cell membranes (to detect both cytoplasmically and luminally disposed proteins) or selectively permeabilized with SLO leaving the ER and Golgi membranes intact (thus allowing detection of only cytoplasmically disposed proteins). Fixed cells were then co-probed with the rabbit anti-E2 antiserum R646 and the anti-c-myc mAb 9E10. The antiserum R646 (raised against the luminally disposed E2 ectodomain) recognized E2 in cells permeabilized with NP-40 but not with SLO (Fig. 5d
). In contrast, the anti-c-myc mAb recognized its epitope in E2p7c-myc in cells permeabilized with both NP-40 and SLO (Fig. 5d
), consistent with the biochemical data above (Fig. 5ac
). Together, these results confirmed that the C-terminal portion of E2p7c-myc is cytoplasmically orientated. Given that this portion of an epitope-tagged p7, when expressed alone, is luminally disposed (Carrere-Kremer et al., 2002
), it seems that p7 is capable of adopting different topologies at various stages in virion morphogenesis.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The conservation of suboptimal cleavage at the E2/p7 site in related pestiviruses has been suggested to indicate a common function (Elbers et al., 1996). Although the E2p7 species in these viruses is not essential for replication (Elbers et al., 1996
; Harada et al., 2000
), it may still play an important role in virus morphogenesis. The results presented here have identified for the first time the formation of a heterodimer between E2p7 and E1, indicating that, like E1E2, the E1E2p7 heterodimer may also play a functional role in virus replication.
The reasons for the complete lack of cleavage at the E2/p7 site in insect cells is at present not clear. The host-derived signal peptidase(s) that cleave HCV structural proteins from the polyprotein remain unidentified. Given that efficient proteolytic processing at the core/E1 and E1/E2 sites occurs in insect cells, it is tempting to speculate that a novel proteinase, not present in Sf cells, may be responsible for cleavage at the E2/p7 site. Identification of the host signal peptidase(s) responsible for cleavage of CE1E2p7 may reveal a mechanism for the peculiar processing events observed.
HCV p7 is an integral membrane protein possessing a double membrane-spanning topology with both of its termini luminally disposed (Carrere-Kremer et al., 2002) (Fig. 6b
). Our investigation into the TM topology of E2p7 by two independent means intriguingly identified a cytoplasmically disposed C-terminal portion of p7 present both in mammalian and insect cells. Taken together, this indicates a dual TM topology for p7 (Fig. 6a and b
). It is not unprecedented for viral membrane proteins to adopt mixed TM topology. For example, the N terminus of HCV NS4B has been reported to assume dual TM topology, with possible distinct functions, on each side of the ER membrane (Lundin et al., 2003
). The hepatitis B virus surface L glycoprotein can act in virus assembly as a matrix-like protein and in virus entry as a receptor-binding protein by adopting different TM topologies (Lambert & Prange, 2001
). Moreover, the Newcastle disease virus fusion protein exists in two topological forms with respect to membranes, one of which has been proposed to be involved in cell-to-cell fusion (McGinnes et al., 2003
).
|
Carrere-Kremer et al. (2004) postulated that the existence of an E2p7 precursor may be a means of keeping p7 inactive in terms of its ion-channel function during glycoprotein maturation and particle assembly or in regulation of cleavage at E2/p7 and/or p7/NS2 cleavage sites. Another possibility is that inefficient cleavage of the E2/p7 site may relate to a mechanism to prevent premature fusion of E2 during virus egress. Similar processing events have been reported in other viruses, including that of the flavivirus prM/E site, whose cleavage is delayed until the stage of virion release, hence preventing E from undergoing acid-catalysed conformational changes during transport of immature virus through the acidic environment of the intermediate compartment (Chambers et al., 1990
; Guirakhoo et al., 1991
, 1992
; Heinz & Allison, 2000
). Inhibition of this cleavage does not prevent infectious virus from being produced, but is thought to be necessary for the production of highly infectious virus particles (Chambers et al., 1990
).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baumert, T. F., Vergalla, J., Satoi, J., Thomson, M., Lechmann, M., Herion, D., Greenberg, H. B., Ito, S. & Liang, T. J. (1999). Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate. Gastroenterology 117, 13971407.[Medline]
Carrere-Kremer, S., Montpellier-Pala, C., Cocquerel, L., Wychowski, C., Penin, F. & Dubuisson, J. (2002). Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 76, 37203730.
Carrere-Kremer, S., Montepellier, C., Lorenzo, L., Brulin, B., Cocquerel, L., Belouzard, S., Penin, F. & Dubuisson, J. (2004). Regulation of hepatitis C virus polyprotein processing by signal peptidase involves determinants at the p7 sequence junctions. J Biol Chem 279, 4138441392.
Chambers, T. J., Hahn, C. S., Galler, R. & Rice, C. M. (1990). Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44, 649688.[CrossRef][Medline]
Clayton, R. F., Owsianka, A., Aitken, J., Graham, S., Bhella, D. & Patel, A. H. (2002). Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles. J Virol 76, 76727682.
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., Op de Beeck, A., Lambot, M., Roussel, J., Delgrange, D., Pillez, A., Wychowski, C., Penin, F. & Dubuisson, J. (2002). Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins. EMBO J 21, 28932902.
Crystal, A. S., Morais, V. A., Pierson, T. C., Pijak, D. S., Carlin, D., Lee, V. M. & Doms, R. W. (2003). Membrane topology of -secretase component PEN-2. J Biol Chem 278, 2011720123.
Dubuisson, J., Hsu, H. H., Cheung, R. C., Greenberg, H. B., Russell, D. G. & Rice, C. M. (1994). Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. J Virol 68, 61476160.[Abstract]
Duvet, S., Cocquerel, L., Pillez, A., Cacan, R., Verbert, A., Moradpour, D., Wychowski, C. & Dubuisson, J. (1998). Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval. J Biol Chem 273, 3208832095.
Elbers, K., Tautz, N., Becher, P., Stoll, D., Rumenapf, T. & Thiel, H. J. (1996). Processing in the pestivirus E2NS2 region: identification of proteins p7 and E2p7. J Virol 70, 41314135.[Abstract]
Evan, G. I., Lewis, G. K., Ramsay, G. & Bishop, J. M. (1985). Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5, 36103616.[Medline]
Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. (1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83, 81228126.[Abstract]
Griffin, S. D., Beales, L. P., Clarke, D. S., Worsfold, O., Evans, S. D., Jaeger, J., Harris, M. P. & Rowlands, D. J. (2003). The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett 535, 3438.[CrossRef][Medline]
Griffin, S. D., Harvey, R., Clarke, D. S., Barclay, W. S., Harris, M. & Rowlands, D. J. (2004). A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria. J Gen Virol 85, 451461.
Guirakhoo, F., Heinz, F. X., Mandl, C. W., Holzmann, H. & Kunz, C. (1991). Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions. J Gen Virol 72, 13231329.[Abstract]
Guirakhoo, F., Bolin, R. A. & Roehrig, J. T. (1992). The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology 191, 921931.[Medline]
Harada, T., Tautz, N. & Thiel, H. J. (2000). E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J Virol 74, 94989506.
Heinz, F. X. & Allison, S. L. (2000). Structures and mechanisms in flavivirus fusion. Adv Virus Res 55, 231269.[Medline]
Lambert, C. & Prange, R. (2001). Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation. J Biol Chem 276, 2226522272.
Lin, C., Lindenbach, B. D., Pragai, B. M., McCourt, D. W. & Rice, C. M. (1994). Processing in the hepatitis C virus E2Ns2 region: identification of P7 and two distinct E2-specific products with different C-termini. J Virol 68, 50635073.[Abstract]
Lindenbach, B. D. & Rice, C. M. (2001). Flaviviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 9911042. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Lundin, M., Monne, M., Widell, A., Von Heijne, G. & Persson, M. A. (2003). Topology of the membrane-associated hepatitis C virus protein NS4B. J Virol 77, 54285438.
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.
McGinnes, L. W., Reitter, J. N., Gravel, K. & Morrison, T. G. (2003). Evidence for mixed membrane topology of the Newcastle disease virus fusion protein. J Virol 77, 19511963.
Mizushima, H., Hijikata, M., Asabe, S., Hirota, M., Kimura, K. & Shimotohno, K. (1994). Two hepatitis C virus glycoprotein E2 products with different C termini. J Virol 68, 62156222.[Abstract]
Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T. & Sato, J. (1982). Growth of human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer Res 42, 38583863.[Abstract]
Owsianka, A., Clayton, R. F., Loomis-Price, L. D., McKeating, J. A. & Patel, A. H. (2001). Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2. J Gen Virol 82, 18771883.
Pavlovic, D., Neville, D. C., Argaud, O., Blumberg, B., Dwek, R. A., Fischer, W. B. & Zitzmann, N. (2003). The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci U S A 100, 61046108.
Pettersson, R. F. (1991). Protein localization and virus assembly at intracellular membranes. Curr Top Microbiol Immunol 170, 67106.[Medline]
Premkumar, A., Wilson, L., Ewart, G. D. & Gage, P. W. (2004). Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride. FEBS Lett 557, 99103.[CrossRef][Medline]
Rose, J. K., Buonocore, L. & Whitt, M. A. (1991). A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques 10, 520525.[Medline]
Rosenberg, S. (2001). Recent advances in the molecular biology of hepatitis C virus. J Mol Biol 313, 451464.[CrossRef][Medline]
Sakai, A., Claire, M. S., Faulk, K., Govindarajan, S., Emerson, S. U., Purcell, R. H. & Bukh, J. (2003). The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences. Proc Natl Acad Sci U S A 100, 1164611651.
Sato, K., Okamoto, H., Aihara, S., Hoshi, Y., Tanaka, T. & Mishiro, S. (1993). Demonstration of sugar moiety on the surface of hepatitis C virions recovered from the circulation of infected humans. Virology 196, 354357.[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]
Takamizawa, A., Mori, C., Fuke, I. & 7 other authors (1991). Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol 65, 11051113.[Medline]
Yagnik, A. T., Lahm, A., Meola, A., Roccasecca, R. M., Ercole, B. B., Nicosia, A. & Tramontano, A. (2000). A model for the hepatitis C virus envelope glycoprotein E2. Proteins 40, 355366.[CrossRef][Medline]
Yamaga, A. K. & Ou, J.-H. (2002). Membrane topology of the hepatitis C virus NS2 protein. J Biol Chem 277, 3322833234.
Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. (1997). Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94, 87388743.
Received 2 November 2004;
accepted 30 November 2004.