Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy1
Author for correspondence: Maria Rapicetta. Fax +39 06 49902662. e-mail rapicett{at}iss.it
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Abstract |
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Introduction |
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The structural region of the HCV genome encodes two proteins, E1 and E2, which constitute the virus envelope glycoproteins (Grakoui et al., 1993 ; Hijikata et al., 1991
). Studies using protein expression systems indicate that E1 and E2 interact to form complexes that are probably the functional envelope subunits of HCV (Deleersnyder et al., 1997
; Dubuisson et al., 1994
; Lanford et al., 1993
; Ralston et al., 1993
). These complexes are retained in the endoplasmic reticulum (ER), where both proteins are modified by N-linked glycosylation (Dubuisson et al., 1994
; Duvet et al., 1998
). A critical role in HCV envelope biogenesis seems to be played by the hydrophobic regions found at the C-terminal ends of both E1 and E2. Indeed, these domains are involved in multiple functions, such as E1 and E2 retention in the ER, membrane anchoring and complex formation (Ciccaglione et al., 1998b
; Cocquerel et al., 1998
, 1999
; Dubuisson, 2000
).
Recently, we reported that expression of E1 in Escherichia coli modifies membrane permeability (Ciccaglione et al., 1998a ). This effect is mainly due to the interaction of a C-terminal hydrophobic region (aa 331383) with the cell membrane (Ciccaglione et al., 2000
), suggesting that insertion of this domain into biological membranes may alter their selectivity to ions or small compounds. Several reports indicate that proteins that form pores in the membrane are equally active in both prokaryotic and eukaryotic cells (Carrasco, 1995
; Dempsey, 1990
). For this reason, the inducible synthesis of proteins in E. coli is a useful system for the identification and analysis of several membrane-active proteins from other animal viruses, such as poliovirus 2B and 3AB (Aldabe et al., 1996
; Lama & Carrasco, 1995
, 1996
), influenza virus M2 (Guinea & Carrasco, 1994
) and human immunodeficiency virus gp41 and Vpu (Arroyo et al., 1995
; Gonzalez & Carrasco, 1998
). In several cases, it has been demonstrated that changes in membrane permeability induced by these proteins are directly related to important alterations in the membrane observed during specific steps of virus infection, such as cell fusion, virus release and glycoprotein biogenesis (Dubay et al., 1992
; Lee et al., 1989
; Doedens & Kirkegaard, 1995
; Henkel et al., 2000
; van Kuppeveld et al., 1997
).
Analysis of membrane-active proteins from virus and non-virus origins indicates that a specific structural organization is required for the formation of pores in the membrane. All these proteins show one or more hydrophobic domains that contain internal, charged hydrophilic residues (Carrasco, 1995 ). Interestingly, we found that a similar structural organization is highly conserved in the internal (aa 262291) and C-terminal (aa 331383) hydrophobic regions of E1 (Bukh et al., 1993
). As E1 has a highly variable amino acid sequence (68·7%), it is likely that these structurally conserved domains could play a specific function during the virus replication cycle.
Alteration of biological membranes by E1 is a potentially important mechanism that may be relevant to studies on HCV biology and for the design of antiviral compounds. In order to demonstrate that this function may be directly related to the intrinsic characteristics of E1, we evaluated the role of the internal hydrophobic domain in modifying membrane permeability in E. coli and analysed the effects that the mutation of conserved amino acids in the C-terminal domain have on membrane-permeabilizing activity.
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Methods |
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Cell lysis assay.
Cell lysis was analysed by measuring cell growth (OD600) at different times post-induction. All data from cell lysis and membrane-permeability assays are the result of at least three independent experiments.
Uridine incorporation into E. coli.
Cells were incubated with 2 µCi/ml [3H]uridine (27·3 Ci/mmol; Amersham) for 2 h before inducing protein expression. Cells were then washed three times with uridine-free growth medium and protein expression was induced as described above. Aliquots of 0·2 ml were pelleted at different times post-induction. To quantify the release of radioactive uridine, supernatants were mixed with L-929 scintillation cocktail (Dupont) and analysed. Radioactivity corresponds to low-molecular-mass compounds that cannot be precipitated by trichloroacetic acid.
Flow cytometry analysis.
Flow cytometry was carried out as described previously (Ciccaglione et al., 1998a ; Arroyo et al., 1995
). Briefly, 8 µl of cells were collected at different times (h) post-induction and stained with 0·005% propidium iodide (PI). Cells were analysed in a FACScan flow cytometer (Becton Dickinson). Each sample for analysis contained from 5000 to 10000 cells.
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Results |
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Lysis of recombinant cultures expressing the internal hydrophobic region
Membrane-active proteins are lytic when expressed in E. coli. Cell lysis is more pronounced when E. coli strains expressing T7 lysozyme, such as E. coli BL21(DE3)pLysS, are used. In this case, the change in membrane permeability caused by the expressed protein may permit the passage of T7 lysozyme into the periplasmic space, where it exerts lytic activity on the bacterial cell wall (Aldabe et al., 1996 ; Arroyo et al., 1995
; Guinea & Carrasco, 1994
; Lama & Carrasco, 1996
). To test the effect of fragments containing the internal hydrophobic region on membrane permeability to T7 lysozyme, we estimated cell lysis by measuring the OD600 of recombinant cultures at different times post-induction (Fig. 2A
). Expression of fragment RH was clearly lytic for E. coli. Cell lysis was also observed in cultures expressing the entire E1 gene. The expression of mutant proteins lacking different fragments of the C-terminal region of E1 produced contrasting results. While synthesis of E340 did not alter cell growth, even after extended times post-induction, the expression of E330 did not influence cell growth in the first hours after induction, but clearly showed a negative effect on the cells if analysed at later times post-induction (Fig. 2A
).
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All together, our findings indicate that RH, which overlaps the internal hydrophobic region of E1, shows membrane-permeabilizing activity in E. coli and could be considered to be an additional membrane-active domain of E1. However, results indicated that not all fragments containing this region are active on membranes. In this regard, analysis of membrane association indicated that E340 was found mainly in the soluble phase of the cell, while E383 and E330 co-purified with the cell membrane (data not shown), suggesting that there may be conformational constraints for the membrane association of E340.
Site-directed mutagenesis of the C-terminal hydrophobic region of E1
The C-terminal hydrophobic region of E1 (aa 331383), unlike the internal one, contains amino acids that are strictly conserved in all HCV genotypes (Bukh et al., 1993 ). Four conserved residues, Arg339, Trp368, Lys370 and Val371, are located close to or inside hydrophobic stretches that show a high propensity to fold into
-helical configuration (Chou & Fasman, 1978
) and have been predicted to be transmembrane (TM) regions (Hofmann & Stoffel, 1993
). It may be possible then that these residues are inserted into the membrane and play a role in pore formation and/or correct membraneprotein interaction. To establish the role of single conserved amino acids in the membrane-permeabilizing activity of E1, we mutated the four conserved amino acids in the C-terminal region of wild-type E1 (Fig. 1
, VP), which is the most membrane-active fragment (Ciccaglione et al., 1998a
). The amino acid substitutions of the VP mutants are indicated in Table 1
. Membrane-permeabilizing activity was analysed by cell lysis and uridine-release assays.
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Uridine release in recombinant cultures expressing VP mutants
The activity of VP mutants on membrane permeability was analysed by measuring the release of [3H]uridine from pre-loaded cultures after treatment with IPTG. As in the cell lysis assay, clones expressing the ArgVal+Lys
Glu and Trp
Arg+Lys
Glu mutations, as well as clones expressing the parental plasmid pET-3a, did not cause the release of uridine (Fig. 3F
). In contrast, radioactive uridine was detected in the supernatant of cultures expressing wild-type VP (Fig. 3C
) and mutants Lys
Ile, Val
Glu, Arg
Val+Lys
Ile (Fig. 3E
) and Trp
Arg (Fig. 3D
). Even in this assay, mutants Lys
Ile, Val
Glu and Arg
Val+Lys
Ile showed an increase in uridine release with respect to the wild-type clone (Fig. 3C
, E
). Finally, the Lys
Glu and Arg
Glu mutants induced a slower release of uridine with respect to the wild-type clone (Fig. 3C
, D
).
According to the results obtained from membrane-permeability assays, analysis of protein expression revealed that VP and all the membrane-active mutants were expressed at a very low level and only for the first hour after induction. This suggests that very low expression of these fragments in E. coli is toxic. In contrast, fragments containing the ArgVal+Lys
Glu and Trp
Arg+Lys
Glu mutations were synthesized at a higher level and their expression had not decreased 4 h after induction (Ciccaglione et al., 1998a
; data not shown).
In conclusion, our findings indicate that clones with the double mutations TrpArg+Lys
Glu and Arg
Val+Lys
Glu lose the ability to make membranes permeable to different compounds and their synthesis is well-tolerated by E. coli, suggesting that the mutated residues could play a crucial role in the membrane activity of E1.
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Discussion |
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In the past, inducible protein expression in E. coli has been used successfully for analysing the membrane-permeabilizing activity of several proteins from other animal viruses (Aldabe et al., 1996 ; Arroyo et al., 1995
; Guinea & Carrasco, 1994
). These studies have demonstrated that the effluence of compounds through the membrane was directly due to an intrinsic ability of these proteins to modify membrane permeability (Lama & Carrasco, 1992
b, 1995
). Consequently, the results obtained by expressing E1 in E. coli can be considered to be a significant indication of a new biochemical function.
The E1 protein of HCV is a type 1 TM glycoprotein with a C-terminal anchor domain. However, computer prediction of a second internal TM domain, in addition to the C-terminal one, indicates that E1 may interact with the membrane through a more complex mechanism involving multiple regions. Our findings show that the RH fragment, which overlaps the internal TM region, is able to modify the permeability of the membrane to different compounds. As permeability changes are detected immediately after inducing RH expression, it is likely that a direct interaction of RH with the membrane may cause an alteration of its integrity.
It has been proposed that the internal hydrophobic region may contain the HCV fusion peptide (Flint et al., 1999 ). Changes in membrane permeability induced by RH indicate that this segment has the ability to disturb the architecture of the lipid bilayer, a property that is intrinsic to viral fusion peptides. Nevertheless, two conserved features of RH, the hydrophobicity and the positively charged ends, as well as the presence of another membrane-active fragment in E1, lead us to favour the hypothesis that considers RH as a domain which co-operates with the C-terminal fragment in modifying membrane permeability. Further studies are, in any case, required to investigate the role of this region in virus fusion.
The hydrophobic region found at the C-terminal end of E1 is 53 amino acids in length (aa 331383). The structure of such a putative TM region is difficult to predict, but it is long enough to traverse the membrane two or three times. Computer analysis reveals the presence of two putative TM segments in this region, which contains the conserved amino acids Arg339, Trp368 and Lys370. Our data demonstrate that these residues are critical for membrane-permeabilizing activity, as mutations affecting these positions abolish the phenotype. It is likely that these amino acids may participate in the formation of the hydrophilic lumen of a membrane pore (Arg and Lys) and/or correctly position the protein into the membrane (Trp), thus allowing permeability.
Recently, it has been reported that Lys370 is required for retaining a chimeric CD4E1 protein in the ER: CD4E1 contains a C-terminal fragment (aa 353383) of E1 fused to a CD4 ectodomain (Cocquerel et al., 2000 ). Our results, reporting that mutations of Lys370 affect membrane activity, support the concept that the C-terminal TM region of E1, whose N-terminal limit still requires a final definition, is involved in more different activities and that specific amino acids, such as Lys370, could play a key role in more than one function. We retain that the exact position that Lys370 assumes, with respect to the membrane environment could be critical for stable association and may influence more E1 activities which are structurally linked to membrane interaction.
The molecular characterization of E1 activity may have important implications for the design of therapeutic agents that specifically block this function. It is interesting to note that amantadine, an antiviral reagent that inhibits influenza virus M2 channel-formation activity, seems to be effective in 18% of patients who previously failed to respond to interferon- therapy (Smith, 1997
). As membrane-permeabilizing activity has been reported only for the E1 protein of HCV, the inhibition of such a function by amantadine is currently under investigation in our laboratory.
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Acknowledgments |
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References |
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Arroyo, J., Boceta, M., González, M. E., Michel, M. & Carrasco, L. (1995). Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41. Journal of Virology 69, 4095-4102.[Abstract]
Bukh, J., Purcell, R. H. & Miller, R. H. (1993). At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide. Proceedings of the National Academy of Sciences, USA 90, 8234-8238.
Carrasco, L. (1995). Modification of membrane permeability by animal viruses. Advances in Virus Research 45, 61-112.[Medline]
Chou, P. Y. & Fasman, G. D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Advances in Enzymology and Related Areas of Molecular Biology 47, 45-148.[Medline]
Ciccaglione, A. R., Marcantonio, C., Costantino, A., Equestre, M., Geraci, A. & Rapicetta, M. (1998a). Hepatitis C virus E1 protein induces modification of membrane permeability in E. coli cells. Virology 250, 1-8.[Medline]
Ciccaglione, A. R., Marcantonio, C., Equestre, M., Jones, I. M. & Rapicetta, M. (1998b). Secretion and purification of HCV E1 protein forms as glutathione S-transferase fusion in the baculovirus insect cell system. Virus Research 55, 157-165.[Medline]
Ciccaglione, A. R., Marcantonio, C., Costantino, A., Equestre, M., Geraci, A. & Rapicetta, M. (2000). Expression and membrane association of hepatitis C virus envelope 1 protein. Virus Genes 21, 223-226.[Medline]
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.
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]
Dempsey, C. E. (1990). The actions of melittin on membranes. Biochimica et Biophysica Acta 1031, 143-161.[Medline]
Doedens, J. R. & Kirkegaard, K. (1995). Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A. EMBO Journal 14, 894-907.[Abstract]
Dubay, J. W., Roberts, S. J., Hahn, B. H. & Hunter, E. (1992). Truncation of the human immunodeficiency virus type 1 transmembrane glycoprotein cytoplasmic domain blocks virus infectivity. Journal of Virology 66, 6616-6625.[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]
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. Journal of Biological Chemistry 273, 32088-32095.
Flint, M., Thomas, J. M., Maidens, C. M., Shotton, C., Levy, S., Barclay, W. S. & McKeating, J. A. (1999). Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. Journal of Virology 73, 6782-6790.
Gonzalez, M. E. & Carrasco, L. (1998). The human immunodeficiency virus type 1 Vpu protein enhances membrane permeability. Biochemistry 37, 13710-13719.[Medline]
Grakoui, A., Wychowski, C., Lin, C., Feinstone, S. M. & Rice, C. M. (1993). Expression and identification of hepatitis C virus polyprotein cleavage products. Journal of Virology 67, 1385-1395.[Abstract]
Guinea, R. & Carrasco, L. (1994). Influenza virus M2 protein modifies membrane permeability in E. coli cells. FEBS Letters 343, 242-246.[Medline]
Henkel, J. R., Gibson, G. A., Poland, P. A., Ellis, M. A., Hughey, R. P. & Weisz, O. A. (2000). Influenza M2 proton channel activity selectively inhibits trans-Golgi network release of apical membrane and secreted proteins in polarized MadinDarby canine kidney cells. Journal of Cellular Biology 148, 495-504.
Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M. & Shimotohno, K. (1991). Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proceedings of the National Academy of Sciences, USA 88, 5547-5551.[Abstract]
Hofmann, K. & Stoffel, W. (1993). TMBASE: a database of membrane spanning protein segments. Hoppe-Seyler.
Hoofnagle, J. H. (1997). Hepatitis C: the clinical spectrum of disease. Hepatology 26, 15-20.
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105-132.[Medline]
Lama, J. & Carrasco, L. (1992). Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A. Journal of Biological Chemistry 267, 15932-15937.
Lama, J. & Carrasco, L. (1995). Mutations in the hydrophobic domain of poliovirus protein 3AB abrogate its permeabilizing activity. FEBS Letters 367, 5-11.[Medline]
Lama, J. & Carrasco, L. (1996). Screening for membrane-permeabilizing mutants of the poliovirus protein 3AB. Journal of General Virology 77, 2109-2119.[Abstract]
Lanford, R. E., Notvall, L., Chavez, D., White, R., Frenzel, G., Simonsen, C. & Kim, J. (1993). Analysis of hepatitis C virus capsid, E1, and E2/NS1 proteins expressed in insect cells. Virology 197, 225-235.[Medline]
Lee, S. J., Hu, W., Fisher, A. G., Looney, D. J., Kao, V. F., Mitsuya, H., Ratner, L. & Wong-Staal, F. (1989). Role of the carboxy-terminal portion of the HIV-1 transmembrane protein in viral transmission and cytopathogenicity. AIDS Research and Human Retroviruses 5, 441-449.[Medline]
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 Virology 67, 6753-6761.[Abstract]
Smith, J. P. (1997). Treatment of chronic hepatitis C with amantadine. Digestive Diseases and Sciences 42, 1681-1687.[Medline]
Studier, F. W. (1991). Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of Molecular Biology 219, 37-44.[Medline]
Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology 189, 113-130.[Medline]
van Kuppeveld, F. J., Hoenderop, J. G., Smeets, R. L., Willems, P. H., Dijkman, H. B., Galama, J. M. & Melchers, W. J. (1997). Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release. EMBO Journal 16, 3519-3532.
Received 9 March 2001;
accepted 17 May 2001.