利用重组丙型肝炎病毒包膜蛋白E2体外检测膜互作和脂质体融合

Belén Yélamos; Mar Rodríguez-Rodríguez; Francisco Gavilanes; Julián Gómez-Gutiérrez

doi:10.21769/BioProtoc.3108

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Peer-reviewed

In vitro Membrane Interaction and Liposome Fusion Assays Using Recombinant Hepatitis C Virus Envelope Protein E2

利用重组丙型肝炎病毒包膜蛋白E2体外检测膜互作和脂质体融合

BY Belén Yélamos

MR Mar Rodríguez-Rodríguez

FG Francisco Gavilanes

JG Julián Gómez-Gutiérrez ^* email

(*contributed equally to this work) 发布: 2018年12月05日第8卷第23期 DOI: 10.21769/BioProtoc.3108 浏览次数: 4935

评审: David PaulJan-Ulrik DahlAli Asghar Kermani

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The authors used this protocol in:

Cover of Biochimica et Biophysica Acta (BBA) - Biomembranes, featuring study using the protocol.

Mar 2018

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Abstract

In order to study the mechanism underlying the Hepatitis C Virus (HCV) fusion process we have performed assays using phospholipid liposomes and a truncated form of E2 protein, E2₆₆₁ (amino acids 384-661 of the HCV polyprotein) lacking the transmembrane region. E2₆₆₁ has been previously generated by using the baculovirus expression system. This form has been used in lipid-protein interaction studies with different model vesicles at different pHs, and monitored using a variety of fluorescent assays. After the analysis of the results, we observed that E2₆₆₁ is able to insert into lipid bilayers and to induce vesicle aggregation, lipid mixing and liposome leakage, showing higher values of membrane destabilization for negatively charged phospholipids at acidic pH. This is indicative of the role of E2 glycoprotein in the HCV initial infective steps, interacting with the target membranes and producing their destabilization.

Keywords: Hepatitis C virus (丙型肝炎病毒)

Envelope proteins E1 and E2 (包膜蛋白E1和E2)

Fusion (融合)

Phospholipid vesicles (磷脂膜泡)

Aggregation (聚集)

Lipid mixing (脂类混合)

Leakage (泄漏)

Background

The fusion of the viral and cellular membranes is the first step of the infective cycle of an enveloped virus (Lemon et al., 2007; Lindenbach et al., 2007). This fusion can take place either at the plasma membrane or in an intracellular location after viral internalization by endocytosis mediated by binding to a receptor located at the plasma membrane. The Hepatitis C virus (HCV) genome is translated into a single large polyprotein, which is processed, among others, into the envelope proteins E1 and E2, which are involved in membrane fusion of this virus with the hepatocyte membranes. E1 (residues 192-383 of the polyprotein) and E2 (residues 384-746) are type-I transmembrane proteins, highly glycosylated, with an N-terminal, water soluble, ectodomain and a C-terminal hydrophobic domain anchoring these glycoproteins to the membrane. Sequence comparisons with fusion proteins of other viruses suggest that E2 could be the fusion protein (Cormier et al., 2004; Rocha-Perugini et al., 2008). However, several studies performed with synthetic peptides suggest the involvement of both E1 and E2 ectodomain sequences in the fusion process. To increase our knowledge of HCV biology two approaches have been developed: the use of HCV pseudoparticles (HCVpp), which consist of E1E2 glycoproteins assembled with retroviral core particles (Lavillette et al., 2006; Meertens et al., 2006), and a more recent cell culture model which enables the propagation of virus in cell culture (HCVcc) (Koutsoudakis et al., 2006). However, these systems are difficult to study fusion in vitro and the results obtained do not indicate which of the two glycoproteins has the fusogenic properties. To advance in the knowledge of HCV fusion mechanism, we have studied the membrane-perturbing properties of the ectodomain of E2, obtained using a baculovirus expression system, by employing a variety of fluorescent assays and different conditions (pH and phospholipid composition). We have previously shown that E2₆₆₁ is folded as an independent unit and is recognized by HCV patient sera antibodies (Rodriguez-Rodriguez et al., 2009). Our study shows that E2₆₆₁ is able to induce the essential steps required for fusion (Rodriguez-Rodriguez et al., 2018).

Materials and Reagents

Pipette tips
Pyrex borosilicate glass tubes (Corning, catalog numbers: 99445-10 [10 x 75 mm] and 99445-13 [13 x 100 mm])
100 nm polycarbonate filters (Whatman, catalog number: 800309)
Absorption and fluorescence cuvettes (Hellma, absorption cuvette 1 x 0.2, catalog number: QS 104-10-40; fluorescence cuvette 1 x 0.2, catalog number: 104002F-10-40; fluorescence cuvette 1 x 0.4, catalog number: 104F-10-40)
Glass column manually made by faculty glass blower facility
Sephadex G-75 (Pharmacia Biotech, catalog number: 17-0051-01)
Chloroform (Scharlau, catalog number: CL02001000)
Methanol (Merck, catalog number: 106002)
Dimyristoylphosphatidylcholine (DMPC) (Avanti Polar Lipids, catalog number: 850345)
Dimyristoylphosphatidylglycerol (DMPG) (Avanti Polar Lipids, catalog number: 840445)
Egg phosphatidylcholine (PC) (Avanti Polar Lipids, catalog number: 840051)
Egg yolk phosphatidylglycerol (PG) (Sigma-Aldrich, catalog number: 50445)
Nitrogen Gas (Carburos metálicos, catalog number: UN1066)
Tris-Base (Sigma-Aldrich, catalog number: T1503)
NaCl (Merck, catalog number: 106404)
2-Morpholinoethanesulfonic acid monohydrate buffer substance MES (Merck, catalog number: 106126)
Sodium citrate (Merck, catalog number: 106448)
EDTA (Sigma-Aldrich, catalog number: 03695)
N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine (NBD-PE) (Avanti Polar Lipids, catalog number: 810143)
N-(lissaminerhodamine B sulfonyl)diacylphosphatidyl-ethanolamine (Rh-PE) (Avanti Polar Lipids, catalog number: 810157)
8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A350)
p-xylenebis-(pyridinium) bromide (DPX) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: X1525)
Liquid nitrogen (Air Liquide, Spain)
Triton X-100 (USB, catalog number: 22686)
Tetrahydrofuran (Sigma-Aldrich, catalog number: 401757)
1,6-diphenyl-1,3,5-hexatriene (DPH) (Sigma-Aldrich, catalog number: D208000)
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) (Sigma-Aldrich, catalog number: T0775)
Medium buffer (see Recipes)

Equipment

Pipettes: volume ranges of 2-20 μl, 20-200 μl and 200-1,000 μl
Speed Vac (Uniequip, model: Univapo 100 H)
Water bath Incubator (Selecta, model: unitronic 320)
Bath sonicator (Bransonic, model: Branson 1200)
Extruder apparatus (Avestin, model: LiposoFast^TM-Basic)
Spectrophotometer (Beckman Coulter, model: Beckman DU-640)
Spectrofluorometer, equipped with a thermostated cell holder in which the temperature of the cuvette is maintained by a circulating water bath (SLM Aminco, model: SLM Aminco 8000C)

Procedure

English

中文翻译

文章信息

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如何引用

Yélamos, B., Rodríguez-Rodríguez, M., Gavilanes, F. and Gómez-Gutiérrez, J. (2018). In vitro Membrane Interaction and Liposome Fusion Assays Using Recombinant Hepatitis C Virus Envelope Protein E2. Bio-protocol 8(23): e3108. DOI: 10.21769/BioProtoc.3108.