MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1
Institut de Biologie et Chimie des Protéines, UPR 412 CNRS, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France2
CNRS-UMR8526, IBL/Institut Pasteur de Lille, 59021 Lille Cedex, France3
University of Reading, School of Animal & Microbial Sciences, PO Box 228, Reading, UK4
Author for correspondence: Arvind Patel. Fax +44 141 337 2236. e-mail a.patel{at}vir.gla.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HCV, a member of the Flaviviridae family, is an enveloped virus with a positive-strand RNA genome of approximately 9·5 kb (Choo et al., 1989 ). The viral genome encodes a single polyprotein of approximately 3010 amino acids (aa) that is processed into functional proteins by host and viral proteases (Houghton, 1996
; Ryan et al., 1998
). The putative HCV structural proteins [core and the two envelope glycoproteins (gp), E1 and E2] are located within the N terminus of the polyprotein, whilst the non-structural proteins reside within the C-terminal part (Clarke, 1997
). Gps E1 and E2, when expressed in vitro, associate to form two types of complexes: a heterodimer stabilized by non-covalent interactions and high molecular mass disulfide-linked aggregates representing misfolded proteins (Choukhi et al., 1998
; Cocquerel et al., 1998
; Deleersnyder et al., 1997
; Dubuisson et al., 1994
; Dubuisson & Rice, 1996
; Ralston et al., 1993
). Both types of complex accumulate within the endoplasmic reticulum (ER), the proposed site for HCV assembly and budding. Due to the lack of a suitable cell culture system for in vitro propagation of HCV, it has been impossible to study the nature of the gp complex present on the virus particle. However, studies using conformation-dependent monoclonal antibodies (MAbs) which specifically recognize nondisulfide-linked E2, either alone or when complexed with E1, strongly suggest that the gp complex stabilized by non-covalent interactions represents the pre-budding form of E1E2 native heterodimers (Deleersnyder et al., 1997
; Michalak et al., 1997
). However disulfide-bridged gp aggregates may have a role in vivo; indeed, Liberman et al. (1999)
reported that ER-retained E2 could activate grp78 (BiP) and grp94 chaperone promoters. Overexpression of grp78 has been reported to decrease the sensitivity of cells to cytotoxic T cell killing, an activity that may be important for HCV persistence. Further work is needed to define possible roles for aggregated E2 in HCV pathogenesis.
HCV gps are thought to initiate infection of the target cells by binding to receptors on the cell plasma membrane followed by membrane fusion and entry. Although the mechanism of HCV entry into cells is unknown, the E2 gp is thought to play a major role in virus attachment to the target cell (Rosa et al., 1996 ). The E2 gp extends from aa 384746 of the polyprotein and it carries regions of extreme hypervariability (Kato et al., 1992
; Mizushima et al., 1994
; Ogata et al., 1991
; Weiner et al., 1991
). The most variable region (HVR-1) is located within the N-terminal 27 residues (aa 384411) of E2, while HVR-2 resides in the 476480 segment. Antibodies specific for epitopes within HVR-1 have been reported to inhibit binding of E2 gp to cells and to block HCV infectivity in vitro and in vivo (Farci et al., 1996
; Habersetzer et al., 1998
; Rosa et al., 1996
; Shimizu et al., 1996
; Zibert et al., 1995
).
The E2 gp carries a C-terminal hydrophobic anchor sequence (aa 718746), removal of which results in secretion of the E2 ectodomain (Matsuura et al., 1994 ; Michalak et al., 1997
; Mizushima et al., 1994
; Selby et al., 1994
; Spaete et al., 1992
). Thus, E2 truncated at either aa 715 and 661 is secreted upon expression in mammalian cells, but only the latter was found to be properly folded (Michalak et al., 1997
). A secreted form of E2 was shown to bind to the surface of Molt-4 cells, and this binding could be blocked by sera from HCV-infected chimpanzees (Rosa et al., 1996
). Furthermore, in chimpanzees vaccinated with recombinant E1E2 gps, protection from challenge virus correlated with the presence of antibodies capable of inhibiting E2 gp binding to cells. The sera were classified as being able to neutralize E2 binding (NOB), leading the authors to postulate that such activity may be used as a surrogate marker for neutralizing antibodies (Rosa et al., 1996
). Using a truncated form of E2, Pileri et al. (1998)
recently identified the cell surface protein, CD81, as a putative receptor for HCV. E2 truncated at aa position 661 (E2661) binds specifically to human but not mouse or African green monkey CD81 (Flint et al., 1999
; Higginbottom et al., 2000
; Pileri et al., 1998
). CD81 is a member of the tetraspanin superfamily of proteins; it has four transmembrane domains and two extracellular loops (EC1 and EC2) (Levy et al., 1998
), the second of which has been shown to bind a truncated form of E2 (Pileri et al., 1998
). MAbs specific for CD81 have been reported to have a variety of effects on cellular processes including proliferation, adhesion and motility (Levy et al., 1998
). Interestingly, we recently demonstrated that E2 engagement of CD81 also influenced cell proliferation and aggregation (Flint et al., 1999
).
There are at least six genotypes of HCV, which differ from each other by 30% over the complete virus genome, with 210% variation between subtypes of the same genotype (Simmonds, 1995 ; Simmonds et al., 1994
). In this study, we have used two closely related genotype 1a strains of HCV, namely Glasgow (Gla), cloned directly from an individual infected with a genotype 1a virus (M. McElwee & R. M Elliott; unpublished), and an infectious cDNA clone of strain H77c (Yanagi et al., 1997
). We compared the antigenic and CD81-binding characteristics of E2660 derived from strains Gla and H77c. We show that in contrast to H77c E2660, Gla protein is predominantly synthesized as misfolded disulfide-linked aggregates and fails to bind CD81. Characterization of several chimeric GlaH77c E2660 gps allowed us to identify regions of E2 important for protein aggregation/folding and for interacting with the putative receptor, CD81.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid constructs.
The E2-encoding cDNA sequences used in this study were derived from two HCV genotype 1a strains, Glasgow (Gla; kindly provided by M. McElwee and R. M. Elliott) and an infectious cDNA clone (pCV-H77c) of strain H77c [a kind gift from Jens Bukh (Yanagi et al., 1997 )]. The nucleotide sequences encoding truncated Gla and H77c E2 from aa 364660 followed by 6x His residues (E2660) were PCR amplified and cloned into mammalian expression vector pCDNA3.1/Zeo(+) (Invitrogen) and the vaccinia virus transfer vector pMJ601 (Davison & Moss, 1990
). The GlaH77c chimeric clones (C1C6) were constructed into pCDNA3.1/Zeo(+) as shown in Fig. 3(A)
. The E2 sequences expressed in all constructs encode the E2 signal sequence (aa 364383), which is cleaved during protein translocation, leaving a mature protein carrying aa 384660 of E2 followed by 6x His (E2660). Nucleotide sequences of all constructs were determined to confirm the origin of inserts. The Gla and H77c E2660 cDNA in pMJ601 was inserted into the thymidine kinase gene of vaccinia virus strain WR by homologous recombination and the recombinant viruses isolated as previously described (Davison & Moss, 1990
).
|
Expression of parental and chimeric E2660.
COS-7 cells were transfected with pCDNA3.1/Zeo(+) alone or with pCDNA3.1/Zeo(+) carrying E2660 sequences using a liposome-mediated method (Rose et al., 1991 ) for 5 h at 37 °C. Cells were washed and incubated in EFC10 for 72 h, after which the extracellular medium was collected and clarified by centrifugation at 3000 r.p.m. for 10 min at 4 °C. For radiolabelling of proteins, the transfected COS-7 cells were incubated at 37 °C for 24 h, washed with PBS, and incubated in methionine-free medium containing 50 µCi/ml [35S]methionine for a further 24 h. The medium of transfected cells was harvested and clarified as described above. To radiolabel E2660 expressed by recombinant vaccinia virus, BHK cells were infected at a multiplicity of 10 p.f.u. per cell and incubated for 5 h at 37 °C. Infected cells were washed with PBS, incubated in methionine-free medium containing 50 µCi/ml [35S]methionine for 18 h and the medium harvested. The E2660 secreted into the medium of transfected or infected cells was quantified using an ELISA-based GNA lectin capture assay as reported previously (Flint et al., 2000
) and described briefly below.
Affinity purification of E2660.
The medium of transfected or infected cells containing [35S]methionine-labelled proteins was equilibrated to 20 mM TrisHCl, pH 7·4, 20 mM iodoacetamide, 150 mM NaCl, 1 mM EDTA, 0·5% Triton X-100 (final concentrations) and incubated with NiNTA (Qiagen) resin for 1 h at 4 °C. The resin was pelleted for 30 s in a microcentrifuge and washed four times with the above buffer. The bound proteins were eluted in wash buffer containing 100 mM EDTA and analysed by SDSPAGE under reducing and non-reducing conditions.
Antibodies.
MAbs AP213 and AP320 were generated in mice immunized with a recombinant form of HCV strain Gla E1E2 expressed in mammalian cells and were epitope mapped by PEPSCAN (unpublished). MAbs H31, H44, H50, H60, H61, H53 and H33 were generated as previously described (Deleersnyder et al., 1997 ). The anti-6x His MAb (6His) was purchased from Qiagen and used according to the manufacturers instructions.
Flow cytometric analysis of E2-cell binding and NOB.
The interaction of E2660 with cells was quantified using a FACS-based assay. In brief, cells under test were washed twice in PBS1%FCS0·05% sodium azide (WB) and resuspended at 2x106/ml; 2x105 cells were incubated with E2 at room temperature for 1 h and unbound antigen was removed by washing twice in WB. Cells were incubated with MAb 6His (1·0 µg/ml) for 1 h at room temperature. Finally, cell-bound MAb was visualized with an anti-mouse IgGPE conjugate (Seralabs) and analysed by FACS (Becton Dickinson). Median fluorescence intensities (MFI) were determined using Cellquest software (Becton Dickinson).
MAbs were evaluated for their ability to inhibit E2 binding to cells as previously reported (Flint et al., 1999 ). Briefly, E2 (at 5 µg/ml) was incubated with the MAb under test (10 µg/ml) for 1 h at room temperature and antigenantibody complex formation was then verified by GNA-capture EIA. Complexes were allowed to bind to cells for 1 h at room temperature, unbound complex was removed by washing twice with WB, and the cell-bound complex was visualized with an anti-species IgGPE conjugate (Seralabs). MFI were determined using Cellquest software and the percentage inhibition determined.
GNA-capture EIA and CD81-capture EIA.
Briefly, GNA (Galanthus nivalis) lectin (Boehringer Mannheim) was used to coat Immulon II EIA plates (Dynal) at 1 µg/ml overnight at 4 °C. After washing in Tris-buffered saline, the plates were blocked with 4% milk powder (Cadburys) and E2 allowed to bind for 2 h at room temperature. Bound antigen was visualized with MAbs specific for E2, an anti-species IgGHRP (Seralabs) and TMB (3,3',5,5'-tetramethylbenzidine; Sigma) substrate. Absorbance values were determined at 450 nm (Dynatech). Purified E2 (gift of M. Houghton, Chiron) was used as a calibrant to enable quantification of E2 levels in transient transfection samples.
GSThCD81 and mCD81 fusion proteins expressing the second extracellular region (EC2) of human or mouse CD81, respectively, were used to coat Immulon II EIA plates (Dynal) at 0·5 µg/ml for 4 h at 37 °C as previously described (Flint et al., 1999 , 2000
). The ability of E2 to specifically bind hCD81 was followed as above for the GNA EIA, using MAbs H53 or 6His to detect CD81-bound E2.
SDSPAGE analysis.
The [35S]methionine-labelled proteins eluted from NiNTA resins were mixed with SDSPAGE denaturation buffer (200 mM TrisHCl, pH 6·7; 0·5% SDS, 10% glycerol) and boiled for 3 min in the presence (reducing conditions) or absence (non-reducing conditions) of -mercaptoethanol. Samples were subjected to SDSPAGE (10% polyacrylamide) and the labelled proteins detected by fluorography.
Sequence analysis.
The protein sequence of the E2 (aa 410660) segment of H77c strain (EMBL accession no. AF011751) was used to search all E2 HCV variants in the EMBL database using the FASTA homology program (Pearson & Lipman, 1988 ). Incomplete sequences were removed from the list of matching sequences and multiple sequence alignment carried out with the CLUSTAL W program (Thompson et al., 1994
). For polymorphic analysis, sequences of isolates known to belong to genotype 1a (i.e. HCV-1, HCV-J1, HCV-H) were used to search the variants reported in the EMBL database. The 1a subtype determination was confirmed by following the recommended standard procedure (Robertson et al., 1998
) and using the representative genotype and subtype sequences reported (Chamberlain et al., 1997
). The EMBL accession numbers for the final set of 11 sequences classified in genotype 1a was as follows: M62382, D10749, M62381, X84079, M62321, M67463, G252549, AF011751, AF011752, AF011753 and AF009606. Sequence analyses were made using the Network Protein Sequence @nalysis facilities (NPS@; Combet et al., 2000
) through the IBCP server (http://pbil.ibcp.fr/NPSA). Visualization and calculation of most represented amino acid at each position were done with the MPSA program (Blanchet et al., 2000
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To further quantify the E2CD81 interaction using a more sensitive assay, we studied the ability of Gla and H77c E2660 to interact with a recombinant form of human CD81, GSThCD81, using an ELISA-based binding assay (Flint et al., 1999 , 2000
). Gla and H77c E2660 gps were tested for their ability to bind GNA lectin (Fig. 2A
), GSTmurineCD81 (mCD81) or GSThCD81 (Fig. 2B
); bound antigen was detected using MAb 6His and anti-mouse IgGHRP. Consistent with our cell-binding data, H77c E2660 bound specifically to GSThCD81 and not GSTmCD81, whereas Gla E2660 failed to interact with either of the CD81 proteins (Fig. 2B
). Similar results were obtained using a number of linear and conformational anti-E2 MAbs to detect the CD81-captured E2660 gps (data not shown).
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several chimeric GlaH77c E2660 gps were generated: all chimeras carrying the aa 525660 region of Gla E2 were produced as high molecular mass aggregates and failed to be recognized by a panel of MAbs. Similar results were obtained with a number of conformation-sensitive E2 specific human MAbs (data not shown). Taken together, these data suggest that amino acids within region 525660 may be important in determining the correct folding and subunit aggregation of the gp. We are currently assessing the effect of mutations within this region in the native E1E2 complex.
Data obtained with chimeric gps which resulted in disulfide-bridged aggregates and which failed to bind conformation-dependent MAbs could not be interpreted further with respect to analysing the regions involved in the CD81 interaction. Substitution of most of the Gla HVR-1 (aa 364406) within H77c (chimera C4) had no significant effect on folding or CD81 binding. However chimera C3, carrying region 384524 of Gla, demonstrated minimal CD81 binding and yet was recognized by all the conformation-dependent MAbs analysed, suggesting that region 384524 may be important in modulating CD81 interaction without affecting protein folding. This conclusion is supported by data from chimera C5, which carries Gla aa 407524 within H77c E2, and which shows no change(s) in antigenic conformation yet fails to show a detectable interaction with CD81 (Fig. 4). Previously, we reported that the CD81-binding site within E2661 is of a conformational nature (Flint et al., 1999
). In addition, MAbs specific for epitopes 480493 and 544551 inhibit the E2CD81 interaction, suggesting that these regions play a direct role in CD81 interaction (Flint et al., 1999
). Our data with GlaH77c E2660 chimeras implicating amino acids in region 407524 in CD81 binding are consistent with and support these observations.
Amino acid sequence comparison analysis between Gla and H77c E2660 shows 88·8% identity (Fig. 5). Since the cysteine residues in Gla and H77c E2660 are completely conserved, it is unlikely that Gla E2660 misfolding originates from aberrant disulfide bond formation, even if the resulting products are intermolecular disulfide-bonded aggregates. Since glycosylation is known to modulate proteinprotein interactions (Gahmberg & Tolvanen, 1996
) and Gla E2660 has two fewer N-linked glycosylation sites (aa positions 476 and 532; Fig. 5
), we focussed our efforts on looking at the effect(s) such changes may have on Gla E2 aggregation and antigenicity. Interestingly, 60% of divergent E2 genotype sequences analysed encode a putative N-glycosylation site at position 476 within HVR-2. However, introduction of G476NG478S and/or D532N in Gla E2660 had no effect on antigenicity or aggregation and protein misfolding (Fig. 6
; data not shown), suggesting that other amino acids may be responsible for the phenotypic differences observed between these two proteins.
Several reports have implicated HVR-1 as having a role in eliciting neutralizing antibodies and as a region responsible for interacting with the cell surface (Farci et al., 1996 ; Rosa et al., 1996
; Shimizu et al., 1996
; Zibert et al., 1995
). Our results do not support a role for the HVR in binding CD81; however, given that CD81 is expressed in a wide range of cell types (Levy et al., 1998
), it is unlikely to be the sole factor conferring liver tropism to HCV (Flint & McKeating, 2000
). It is possible that other candidate receptor(s) for HCV exist (Agnello et al., 1999
) and that involvement of HVR-1 in E2 binding the cell surface via such factors cannot be ruled out.
Our unpublished data show that the full-length E2 found in the H77c E1E2 native (but not aggregated) complex binds CD81. This result, together with previously published data, suggest that H77c E2660 adopts a conformation that is similar to that found in the native E1E2 complex. We have recently generated a number of GlaH77c chimeras in the context of full-length E1E2 glycoproteins to further delineate the role of amino acids involved in E1E2 complex formation and its subsequent interaction with CD81. Site-directed mutagenesis of critical amino acid residues identified in this study will increase our understanding of the mechanisms involved in these processes.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blanchet, C., Combet, C., Geourjon, C. & Deleage, G.(2000). MPSA: Integrated system for multiple protein sequence analysis with client/server capabilities.Bioinformatics16, 286-288.[Abstract]
Chamberlain, R. W., Adams, N. J., Taylor, L. A., Simmonds, P. & Elliott, R. M.(1997). The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa.Biochemical and Biophysical Research Communications236, 44-49.[Medline]
Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M.(1989). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral-hepatitis genome. Science244, 359-362.[Medline]
Choukhi, A., Ung, S., Wychowski, C. & Dubuisson, J.(1998). Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins. Journal of Virology72, 3851-3858.
Clarke, B.(1997). Molecular virology of hepatitis C virus.Journal of General Virology78, 2397-2410.
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. Journal of Virology72, 2183-2191.
Combet, C., Blanchet, C., Geourjon, C. & Deleage, G.(2000). NPS@: network protein sequence analysis.Trends in Biochemical Sciences25, 147-150.[Medline]
Davison, A. J. & Moss, B.(1990). New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins.Nucleic Acids Research18, 4285-4286.[Medline]
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.Journal of Virology71, 697-704.[Abstract]
Dubuisson, J. & Rice, C. M.(1996). Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin.Journal of Virology70, 778-786.[Abstract]
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.Journal of Virology68, 6147-6160.[Abstract]
Farci, P., Shimoda, A., Wong, D., Cabezon, T., DeGioannis, D., Strazzera, A., Shimizu, Y., Shapiro, M., Alter, H. J. & Purcell, R. H.(1996). Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein.Proceedings of the National Academy of Sciences, USA93, 15394-15399.
Flint, M. & McKeating, J. A. (2000). The role of hepatitis C virus glycoproteins in infection. Medical Virology (in press).
Flint, M., Maidens, C., Loomis-Price, L. D., Shotton, C., Dubuisson, J., Monk, P., Higginbottom, A., Levy, S. & McKeating, J. A.(1999). Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81.Journal of Virology73, 6235-6244.
Flint, M., Dubuisson, J., Maidens, C., Harrop, R., Guile, G. R., Borrow, P. & McKeating, J. A.(2000). Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein.Journal of Virology74, 702-709.
Gahmberg, C. G. & Tolvanen, M. (1996). Why mammalian cell surface proteins are glycoproteins. Trends in Biochemical Sciences 21, 308311; erratum 21, 491.[Medline]
Habersetzer, F., Fournillier, A., Dubuisson, J., Rosa, D., Abrignani, S., Wychowski, C., Nakano, I., Trepo, C., Desgranges, C. & Inchauspe, G.(1998). Characterization of human monoclonal antibodies specific to the hepatitis C virus glycoprotein E2 with in vitro binding neutralization properties.Virology249, 32-41.[Medline]
Higginbottom, A., Quinn, E. R., Kuo, C.-C., Flint, M., Wilson, L., Pessi, A., Nicosia, A., Monk, P., McKeating, J. A. & Levy, S.(2000). Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2.Journal of Virology74, 3642-3649.
Houghton, M. (1996). Hepatitis C viruses. In Fields Virology, 3rd edn, pp. 10351058. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Kato, N., Ootsuyama, Y., Tanaka, T., Nakagawa, M., Nakazawa, T., Muraiso, K., Ohkoshi, S., Hijikata, M. & Shimotohno, K.(1992). Marked sequence diversity in the putative envelope proteins of hepatitis C viruses.Virus Research22, 107-123.[Medline]
Kolykhalov, A. A., Feinstone, S. M. & Rice, C. M.(1996). Identification of a highly conserved sequence element at the 3' terminus of hepatitis C virus genome RNA.Journal of Virology70, 3363-3371.[Abstract]
Lavanchy, D., Purcell, R., Hollinger, F. B., Howard, C., Alberti, A., Kew, M., Dusheiko, G., Alter, M., Ayoola, E., Beutels, P., Bloomer, R., Ferret, B., Decker, R., Esteban, R., Fay, O., Fields, H., Fuller, E. C., Grob, P., Houghton, M., Leung, N., Locarnini, S. A., Margolis, H., Meheus, A., Miyamura, T., Mohamed, M. K., Tandon, B., Thomas, D., Head, H. T., Toukan, A. U., VanDamme, P., Zanetti, A., Arthur, R., Couper, M., Damelio, R., Emmanuel, J. C., Esteves, K., Gavinio, P., Griffiths, E., Hallaj, Z., Heuck, C. C., Heymann, D. L., Holck, S. E., Kane, M., Martinez, L. J., Meslin, F., Mochny, I. S., Ndikuyeze, A., Padilla, A. M., Rodier, G. R. M., Roure, C., Savage, F. & Vercauteren, G.(1999). Global surveillance and control of hepatitis C.Journal of Viral Hepatitis6, 35-47.[Medline]
Levy, S., Todd, S. C. & Maecker, H. T.(1998). CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system.Annual Review of Immunology16, 89-109.[Medline]
Liberman, E., Fong, Y. L., Selby, M. J., Choo, Q. L., Cousens, L., Houghton, M. & Yen, T. S. B.(1999). Activation of the grp78 and grp94 promoters by hepatitis C virus E2 envelope protein.Journal of Virology73, 3718-3722.
Matsuura, Y., Suzuki, T., Suzuki, R., Sato, M., Aizaki, H., Saito, I. & Miyamura, T.(1994). Processing of E1 and E2 glycoproteins of hepatitis C virus expressed in mammalian and insect cells.Virology205, 141-150.[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.Journal of General Virology78, 2299-2306.[Abstract]
Mizushima, H., Hijikata, M., Asabe, S., Hirota, M., Kimura, K. & Shimotohno, K.(1994). Two hepatitis C virus glycoprotein E2 products with different C termini.Journal of Virology68, 6215-6222.[Abstract]
Ogata, N., Alter, H. J., Miller, R. H. & Purcell, R. H.(1991). Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proceedings of the National Academy of Sciences, USA88, 3392-3396.[Abstract]
Patel, J., Patel, A. H. & McLauchlan, J.(1999). Covalent interactions are not required to permit or stabilize the non-covalent association of hepatitis C virus glycoproteins E1 and E2.Journal of General Virology80, 1681-1690.[Abstract]
Pearson, W. R. & Lipman, D. J.(1988). Improved tools for biological sequence comparison.Proceedings of the National Academy of Sciences, USA85, 2444-2448.[Abstract]
Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D., Grandi, G. & Abrignani, S.(1998). Binding of hepatitis C virus to CD81.Science282, 938-941.
Ralston, R., Thudium, K., Berger, K., Kuo, C., Gervase, B., Hall, J., Selby, M., Kuo, G., Houghton, M. & Choo, Q. L.(1993). Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.Journal of Virology67, 6753-6761.[Abstract]
Robertson, B., Myers, G., Howard, C., Brettin, T., Bukh, J., Gaschen, B., Gojobori, T., Maertens, G., Mizokami, M., Nainan, O., Netesov, S., Nishioka, K., Shini, T., Simmonds, P., Smith, D., Stuyver, L. & Weiner, A.(1998). Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization.Archives of Virology143, 2493-2503.[Medline]
Rosa, D., Campagnoli, S., Moretto, C., Guenzi, E., Cousens, L., Chin, M., Dong, C., Weiner, A. J., Lau, J. Y. N., Choo, Q. L., Chien, D., Pileri, P., Houghton, M. & Abrignani, S.(1996). A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proceedings of the National Academy of Sciences, USA93, 1759-1763.
Rose, J. K., Buonocore, L. & Whitt, M. A.(1991). A new cationic liposome reagent mediating nearly quantitative transfection of animal cells.Biotechniques10, 520-525.[Medline]
Ryan, M. D., Monaghan, S. & Flint, M.(1998). Virus-encoded proteinases of the Flaviviridae.Journal of General Virology79, 947-959.
Selby, M. J., Glazer, E., Masiarz, F. & Houghton, M.(1994). Complex processing and proteinprotein Interactions in the E2Ns2 region of HCV. Virology204, 114-122.[Medline]
Shimizu, Y. K., Igarashi, H., Kiyohara, T., Cabezon, T., Farci, P., Purcell, R. H. & Yoshikura, H.(1996). A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures.Virology223, 409-412.[Medline]
Simmonds, P.(1995). Variability of hepatitis C virus. Hepatology21, 570-583.[Medline]
Simmonds, P., Alberti, A., Alter, H. J., Bonino, F., Bradley, D. W., Brechot, C., Brouwer, J. T., Chan, S. W., Chayama, K., Chen, D. S., Choo, Q. L., Colombo, M., Cuypers, H. T. M., Date, T., Dusheiko, G. M., Esteban, J. I., Fay, O., Hadziyannis, S. J., Han, J., Hatzakis, A., Holmes, E. C., Hotta, H., Houghton, M., Irvine, B., Kohara, M., Kolberg, J. A., Kuo, G., Lau, J. Y. N., Lelie, P. N., Maertens, G., McOmish, F., Miyamura, T., Mizokami, M., Nomoto, A., Prince, A. M., Reesink, H. W., Rice, C., Roggendorf, M., Schalm, S. W., Shikata, T., Shimotohno, K., Stuyver, L., Trepo, C., Weiner, A., Yap, P. L. & Urdea, M. S.(1994). A proposed system for the nomenclature of hepatitis C viral genotypes.Hepatology19, 1321-1324.[Medline]
Spaete, R. R., Alexander, D., Rugroden, M. E., Choo, Q. L., Berger, K., Crawford, K., Kuo, C., Leng, S., Lee, C., Ralston, R. and others (1992). Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells. Virology 188, 819830.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Research22, 4673-4680.[Abstract]
Weiner, A. J., Brauer, M. J., Rosenblatt, J., Richman, K. H., Tung, J., Crawford, K., Bonino, F., Saracco, G., Choo, Q. L., Houghton, M. & Han, J. H.(1991). Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and Ns1 proteins and the pestivirus envelope glycoproteins.Virology180, 842-848.[Medline]
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.Proceedings of the National Academy of Sciences, USA94, 8738-8743.
Zibert, A., Schreier, E. & Roggendorf, M.(1995). Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment.Virology208, 653-663.[Medline]
Received 31 May 2000;
accepted 11 August 2000.