Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas, Sofias Avenue, Athens, Greece1
Centre de Genetique Moleculaire et Cellulaire, UMR 5534 CNRS, Universite Claude Bernard Lyon I, 69622 Villeurbanne Cedex, France2
Section of Microbiology, University of Ferrara, Via Luigi Borsari 46, Ferrara 1-44100, Italy3
Author for correspondence: Penelope Mavromara. Fax +30 1 647 88 77. e-mail penelopm{at}hol.gr
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Abstract |
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Main text |
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HCV E1 and E2 are heavily glycosylated type I transmembrane proteins. A hydrophobic domain at their carboxy terminus acts as a retention signal for the endoplasmic reticulum (ER) and is required for the correct assembly of the two glycoproteins (Cocquerel et al., 1998 , 1999
, 2000
; Dubuisson, 2000
; Flint & McKeating, 1999
; Patel et al., 2001
). A number of independent studies have shown that E1 and E2 interact to form two types of complexes. One type consists of non-covalently associated E1/E2 heterodimers and is believed to result from the productive folding and assembly of the two glycoproteins (Deleersnyder et al., 1997
; Michalak et al., 1997
). The other type consists of disulfide-linked E1/E2 heterodimers, which fail to acquire their correct conformation and form aggregates that show prolonged association with ER chaperones (Choukhi et al., 1998
, 1999
; Deleersnyder et al., 1997
; Dubuisson et al., 1994
). Interestingly, recent studies suggest that both types of E1E2 complex may actually occur in vivo and may play distinct roles in the life cycle and pathogenesis of the virus (Liberman et al., 1999
).
To date, despite the many efforts, an efficient tissue culture system for the propagation of HCV is still not available. Thus, studies on HCV protein structure and function rely on the use of heterologous expression systems. Herpes simplex virus type 1 (HSV-1) amplicons represent unique virus expression vectors because their genome comprises multiple copies of plasmid DNA. The amplicon plasmid contains one copy of an HSV-1 origin of replication (usually ori-S), a packaging signal sequence (pac), which is contained within the repeated sequence of the HSV genome, and the transgene (Freese et al., 1990
; Frenkel et al., 1994
; Spaete & Frenkel, 1982
, 1985
). In the presence of HSV-1 helper virus, the plasmid DNA is amplified (presumably by a rolling-circle mechanism) into a head-to-tail concatamer, which is then packaged into defective HSV-1 particles that are up to one genome size (
150 kb) (Kwong & Frenkel, 1984
). Amplicons have been employed successfully as vectors for the transfer of a variety of genes of neurobiological or therapeutical interest, as well as genes encoding proteins from heterologous virus families (Savard et al., 1997
; Sena-Esteves et al., 1999
, 2000
; Costantini et al., 1999
). HSV-1-based amplicon vectors combine a number of features that make them attractive virus vectors for the expression of heterologous genes. These include the ability to efficiently infect various cell lines, the ability to carry high copy numbers of the transgene and the simplicity of constructing these vectors. Most importantly, new strategies have been developed recently, allowing the generation of either limited amounts of helper-free amplicon vectors (Saeki et al., 2001
) or large amounts of vector stocks presenting a high amplicon to non-pathogenic helper virus ratio (Logvinoff & Epstein, 2001
), thus providing safe virus vectors for vaccine development.
The goal of this study was to explore the potential of HSV-1-based amplicon vectors as alternative expression systems for the study of HCV envelope glycoproteins. For this purpose, the regions encoding the E1E2p7 (aa 191807) or E2p7 (aa 383807) polypeptides, amplified by PCR from a plasmid vector containing the cDNA sequence of HCV-1a (H) (kindly provided by G. Inchauspe, INSERM, Lyon, France), were cloned into the pA-SKlacZ amplicon plasmid under the control of the HSV-1 IE4 (22/
47) promoter. To ensure efficient processing of the HCV glycoproteins in the context of the HSV-1-based vectors, the signal sequences of E1 in the E1E2p7 construct and E2 in the E2p7 construct have been substituted by the signal sequence of the HSV-1 glycoprotein E (gE) (nt 141183141309), corresponding to wild-type HSV-1 (F). The sequence of gE was obtained from plasmid pHPI400 by PCR (Miriagou et al., 1995
). pA-SKlacZ is a pBluescript II plasmid (Stratagene), which contains the amplicon module (ori-S and
sequences) from the pA-SF1 plasmid (Lowenstein et al., 1994
) and also a LacZ/
-galactosidase expression cassette [based on the human cytomegalovirus (HCMV) immediate-early promoter and the simian virus type 40 poly(A) sequences, kindly provided by P. Lowenstein, University of Manchester, Manchester, UK] (Fig. 1a
).
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To assess initially the ability of HSV-1-based amplicon vectors to express the HCV glycoproteins, HepG2 cells (ATCC) were infected with pA-SK lacZE1E2p7 or pA-SK lacZE2p7 amplicon vectors at an m.o.i. of 1 and an A:H ratio of 1 for both stocks. Cells were lysed at different times post-infection (p.i.) in TBS buffer (10 mM TrisHCl pH 7·5, 150 mM NaCl and 2 mM EDTA) containing 0·5% Igepal CA630 (Sigma), 0·1 mM PMSF and 20 mM iodocetamide. Cell lysates were analysed by SDSPAGE and immunoblot analysis using anti-E2, anti-E1 (kindly provided by J. Dubuisson, Pasteur Lille, Lille, France) or anti-
-gal (Gibco BRL) monoclonal antibodies (mAbs). As shown in Fig. 1(b)
, both amplicon vectors supported efficient expression of E2 (6668 kDa) and E1 (3135 kDa) proteins. Similar results were obtained with amplicon stocks with A:H ratios of 100, prepared following infection of TE-CRE30 cells (data not shown). Consistent with previous observations, the E2 protein from the E1E2p7-expressing amplicon vector resolved into two bands, which probably arise from inefficient cleavage of the E2p7 site (Dubuisson et al., 1994
). Notably, in the case of the pA-SK lacZE2p7 amplicon vector, the expression levels of E2 were repeatedly lower and only the faster migrating band was the major E2 protein observed after 15 h p.i. Since similar amounts of
-galactosidase were produced by the two vectors [Fig. 1b
, (i) and (iv)], the possibility of an intrinsic problem of the amplicon vector stock was unlikely, suggesting that the presence of upstream nucleotide sequences and/or the presence of E1 may affect the processing and the levels of E2 expressed in this system.
Secondly, in order to assess the levels of expression obtained from the amplicon vectors, we analysed the expression of E1 and E2 in cells infected in parallel with the pA-SK lacZE1E2p7 amplicon vector or with the replication-competent recombinant HSV-1 rHPI A2/E1E2p7 virus. This virus contains identical E1E2p7-coding sequences expressed from the strong chimeric 1 promoter (kindly provided by B. Roizman, University of Chicago, Chicago, USA) and was generated by homologous recombination between wild-type HSV-1(F) viral DNA and a plasmid shuttle vector (U. Georgopoulou, A. Caravokiri and P. Mavromara; unpublished data) containing the HCV sequences flanked by HSV-1 thymidine kinase homologous sequences, as described previously (Post & Roizman, 1981
). The chimeric
1 promoter was designed to combine the potency of an HSV-1
promoter and the ability of long-term expression of a
1 promoter. Infections were performed in the presence of phosphonoacetic acid (PAA) (300 µg/ml). Under these conditions, viral DNA replication is inhibited and expression from the
1 HSV-1 promoter is reduced significantly (Roizman, 1996
). Therefore, the activity of the
1 promoter would be due primarily to its
component. This allows an indirect comparison between the two expression systems. As shown in Fig. 1[c
, (i) and (ii)], Vero cells infected with the pA-SK lacZE1E2p7 amplicon vector at an m.o.i. of 0·3 supported higher levels of E1 and E2 expression than cells infected with the HSV-1 rHPI A2/E1E2p7 recombinant virus at an m.o.i. of 3. This result suggests that the amplicon vectors efficiently produce high levels of the HCV glycoproteins.
Finally, to investigate the long-term kinetics of expression by the amplicon vectors, we performed a pulse-labelling experiment to detect newly synthesized E2 at several h p.i. In order to avoid CPE of the infected cells due to the replication of helper virus, the experiment was performed in the presence of PAA. Vero cell monolayers cultured in 25 cm2 flasks were infected with the pA-SK LacZE1E2p7 amplicon vector at an m.o.i. of 2·5. Cells were labelled with 100 µCi/ml S35-trans label (ICN) for 2 h before harvesting at the different times p.i. The cell lysates were then immunoprecipitated with the anti-E2 mAb. After immunoprecipitation, the proteins were analysed by SDSPAGE, transferred to a nitrocellulose membrane and subjected to autoradiography. We found that E2 was produced even after 50 h p.i. (Fig. 1d), providing evidence for the long-term expression of the HCV glycoproteins in the HSV-1 amplicon-infected cells.
Previous studies have shown that the HCV E1 and E2 glycoproteins, when expressed in mammalian cells, are retained in the ER and remain sensitive to endo-N-acetylglucosaminidase H (EndoH). Notably, all available data indicate that, under these conditions, the majority of E1 and E2 proteins have the tendency for aberrant disulfide bond formation, while the efficiency of non-covalently associated E1/E2 heterodimers is low (Deleersnyder et al., 1997 ; Michalak et al., 1997
; Patel et al., 1999
). In order to study the behaviour of E1 and E2 in the context of the HSV-1-based vectors, we performed three series of experiments. Initially, the sensitivity of E1 and E2 to the EndoH and N-glycosidase F (PNGaseF) endoglycosidases was studied in cells infected with the pA-SK lacZE1E2p7 vector. Sensitivity of a glycoprotein to EndoH treatment indicates that the protein is resident in the ER or the cis-Golgi and does not migrate further in the secretory pathway. Infected WRL 68 cells (kindly provided by A. Budkowska) or Vero cells were labelled from 2 to 18 h p.i. with 30 µCi/ml S35-trans label and cell lysates were immunoprecipitated, as described previously by Dubuisson et al. (1994)
, using the anti-E2 mAb. Immunoprecipitates were divided in three aliquots and were digested subsequently with either EndoH or PNGaseF (New England Biolabs), according to the manufacturers protocol, or left untreated. Samples were analysed by SDSPAGE and autoradiography [Fig. 2a
, (i)]. The presence of E1 and E2 in the immunoprecipitated materials was confirmed by immunoblot analysis using the anti-E1 and anti-E2 mAbs [Fig. 2a
, (ii) and (iii)]. As shown in Fig. 2(a)
, the patterns observed with EndoH or PNGaseF were essentially the same for both glycoproteins, indicating that all N-linked oligosaccharides were of the immature form, indicative of the retention of the protein in the ER. We obtained similar results with the HSV-1 recombinant rHPI A2/E1E2p7 virus (data not shown). The ER localization of E1 and E2 expressed by the amplicon vectors and the recombinant virus was also verified by indirect immunofluorescence. HepG2 cells were infected with the pA-SKlacZE1E2p7 amplicon vector at an m.o.i. of 0·5. At 6 h p.i., cells were fixed with 4% paraformaldehyde, permeabilized with 0·1% Triton X-100 and labelled with the anti-E1 (Fig. 2b
, panels c and i) or anti-E2 (Fig. 2b
, panels f and l) mAbs, followed by goat anti-mouse antibody conjugated to Alexa fluor 488 (green) (Molecular Probes). For ER-staining, the anti-ER polyclonal antibody (Fig. 2b
, panels b, e, h and k), followed by goat anti-rabbit antibody conjugated to Alexa fluor 568 (red) (Molecular Probes) was used. E1 and E2 gave a fine, reticular, ER-like pattern of localization that was confirmed further by extensive co-localization with an ER marker (anti-ER rabbit polyclonal antibody, kindly provided by E. Coudrier, Institute Curie Paris, Paris, France) (Fig. 2b
, panels a and d). We conclude, therefore, that E1 and E2 expressed by the HSV-1-based vectors are processed correctly by the host peptidases and show the expected patterns of intracellular localization and post-translational glycosylation.
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Acknowledgments |
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References |
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Choukhi, A., Pillez, A., Drobecq, H., Sergheraert, C., Wychowski, C. & Dubuisson, J. (1999). Characterization of aggregates of hepatitis C virus glycoproteins. Journal of General Virology 80, 3099-3107.
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 Virology 72, 2183-2191.
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. Journal of Virology 73, 2641-2649.
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. Journal of Virology 74, 3623-3633.
Costantini, L. C., Jacoby, D. R., Wang, S., Fraefel, C., Breakefield, X. O. & Isacson, O. (1999). Gene transfer to the nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors. Human Gene Therapy 10, 2481-2494.[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 Virology 71, 697-704.[Abstract]
Dubuisson, J. (2000). Folding, assembly and subcellular localization of hepatitis C virus glycoproteins. Current Topics in Microbiology and Immunology 242, 135-148.[Medline]
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 Virology 68, 6147-6160.[Abstract]
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. Journal of Biological Chemistry 275, 30605-30609.
Flint, M. & McKeating, J. A. (1999). The C-terminal region of the hepatitis C virus E1 glycoprotein confers localization within the endoplasmic reticulum. Journal of General Virology 80, 1943-1947.
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 Virology 74, 702-709.
Francki, R. I. B., Fauquet, C. M., Knudson, D. L. & Brown. F. (editors) (1991). Classification and nomenclature of viruses. Fifth Report of the International Committee on the Taxonomy of Viruses. New York: SpringerVerlag.
Freese, A., Geller, A. I. & Neve, R. (1990). HSV-1 vector mediated neuronal gene delivery. Strategies for molecular neuroscience and neurology. Biochemical Pharmacology 40, 2189-2199.[Medline]
Frenkel, N., Singer, O. & Kwong, A. D. (1994). The herpes simplex virus amplicon: a versatile defective virus vector. Gene Therapy 1, S40-S46.[Medline]
Hoofnagle, J. H. (1997). Hepatitis C: the clinical spectrum of disease. Hepatology 26, 15S-20S.[Medline]
Kwong, A. D. & Frenkel, N. (1984). Herpes simplex virus amplicon: effect of size on replication of constructed defective genomes containing eucaryotic DNA sequences. Journal of Virology 51, 595-603.[Medline]
Liberman, E., Fong, Y. L., Selby, M. J., Choo, Q. L., Cousens, L., Houghton, M. & Yen, T. S. (1999). Activation of the grp78 and grp94 promoters by hepatitis C virus E2 envelope protein. Journal of Virology 73, 3718-3722.
Logvinoff, C. & Epstein, A. L. (2000). Intracellular Cre-mediated deletion of the unique packaging signal carried by a herpes simplex virus type 1 recombinant and its relationship to the cleavage-packaging process. Journal of Virology 74, 8402-8412.
Logvinoff, C. & Epstein, A. L. (2001). A novel approach for herpes simplex virus type 1 amplicon vector production, using the Cre-loxP recombination system to remove helper virus. Human Gene Therapy 20, 161-167.
Lowenstein, P. R., Fournel, S., Bain, D., Tomasec, P., Clissold, P., Castro, M. G. & Epstein, A. L. (1994). Herpes simplex virus 1 (HSV-1) helper co-infection affects the distribution of an amplicon encoded protein in glia. Neuroreport 5, 1625-1630.[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 Virology 78, 2299-2306.[Abstract]
Miriagou, V., Argnani, R., Kakkanas, A., Georgopoulou, U., Manservigi, R. & Mavromara, P. (1995). Expression of the herpes simplex virus type 1 glycoprotein E in human cells and in Escherichia coli: protection studies against lethal viral infection in mice. Journal of General Virology 76, 3137-3143.[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 Virology 80, 1681-1690.[Abstract]
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, 58-68.[Medline]
Post, L. E. & Roizman, B. (1981). A generalized technique for deletion of specific genes in large genomes: gene 22 of herpes simplex virus 1 is not essential for growth. Cell 25, 227-232.[Medline]
Reed, K. E. & Rice, C. M. (2000). Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Current Topics in Microbiology and Immunology 242, 55-84.[Medline]
Roizman, B. (1996). The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors. Proceedings of the National Academy of Sciences, USA 93, 11307-11312.
Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, X. O. & Chiocca, E. A. (2001). Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome. Molecular Therapy 3, 591-601.[Medline]
Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., Ohta, Y. and others (1990). Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proceedings of the National Academy of Sciences, USA 87, 65476549.[Abstract]
Savard, N., Cosset, F. L. & Epstein, A. L. (1997). Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors. Journal of Virology 71, 4111-4117.[Abstract]
Sena-Esteves, M., Saeki, Y., Camp, S. M., Chiocca, E. A. & Breakefield, X. O. (1999). Single-step conversion of cells to retrovirus vector producers with herpes simplex virusEpsteinBarr virus hybrid amplicons. Journal of Virology 73, 10426-10439.
Sena-Esteves, M., Saeki, Y., Fraefel, C. & Breakefield, X. O. (2000). HSV-1 amplicon vector: simplicity and versatility. Molecular Therapy 2, 9-15.[Medline]
Spaete, R. R. & Frenkel, N. (1982). The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector. Cell 30, 295-304.[Medline]
Spaete, R. R. & Frenkel, N. (1985). The herpes simplex virus amplicon: analyses of cis-acting replication functions. Proceedings of the National Academy of Sciences, USA 82, 694-698.[Abstract]
Received 24 August 2001;
accepted 8 November 2001.