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• W2142582216 abstract "Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry. Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry. Hepatitis C virus (HCV) 4The abbreviations used are: HCVhepatitis C virustmdtransmembrane domainHCVppHCV pseudoparticlesHCVcccell culture produced HCVFFUfocus forming unit. is an important public health concern worldwide, as it is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped virus that belongs to the Hepacivirus genus of the Flaviviridae family (1Lindenbach B.D. Thiel H.J. Rice C.M. Flaviviridae: The Viruses and Their Replication. Vol. 1. D. M. Knipe and P. M. Howley, Philadelphia2007: 1001-1003Google Scholar). The two surface glycoproteins, E1 and E2, are processed by signal peptidases of the endoplasmic reticulum from a 3000-amino acid-long polyprotein encoded by the HCV genome (2Penin F. Dubuisson J. Rey F.A. Moradpour D. Pawlotsky J.M. Hepatology. 2004; 39: 5-19Crossref PubMed Scopus (514) Google Scholar). hepatitis C virus transmembrane domain HCV pseudoparticles cell culture produced HCV focus forming unit. Because of difficulties in propagating HCV in cell culture, many gaps remain in our understanding of the functions of E1 and E2. A major advance in the investigation of their functions was the development of HCV pseudoparticles (HCVpp) consisting of native HCV envelope glycoproteins E1 and E2 assembled onto retroviral core particles (3Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (944) Google Scholar, 4Drummer H.E. Maerz A. Poumbourios P. FEBS Lett. 2003; 546: 385-390Crossref PubMed Scopus (169) Google Scholar, 5Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C.M. McKeating J.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 7271-7276Crossref PubMed Scopus (687) Google Scholar). Extensive characterization of HCVpp showed that they mimic the early steps of the HCV life cycle (6Bartosch B. Cosset F.L. Virology. 2006; 348: 1-12Crossref PubMed Scopus (135) Google Scholar, 7Cocquerel L. Voisset C. Dubuisson J. J. Gen. Virol. 2006; 87: 1075-1084Crossref PubMed Scopus (161) Google Scholar). Furthermore, data obtained with HCVpp can now also be confirmed with the developed cell culture system that allows efficient amplification of HCV (HCVcc) (8Lindenbach B.D. Evans M.J. Syder A.J. Wölk B. Tellinghuisen T.L. Liu C.C. Maruyama T. Hynes R.O. Burton D.R. McKeating J.A. Rice C.M. Science. 2005; 309: 623-626Crossref PubMed Scopus (1949) Google Scholar, 9Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Kräusslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Nat. Med. 2005; 11: 791-796Crossref PubMed Scopus (2409) Google Scholar, 10Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. Wieland S.F. Uprichard S.L. Wakita T. Chisari F.V. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 9294-9299Crossref PubMed Scopus (1513) Google Scholar). The E1 (31 kDa) and E2 (70 kDa) proteins are glycosylated in their large N-terminal ectodomains and are anchored into the membrane by their C-terminal transmembrane domains. E1 and E2 form a heterodimer stabilized by noncovalent interactions that is retained in the endoplasmic reticulum (11Deleersnyder V. Pillez A. Wychowski C. Blight K. Xu J. Hahn Y.S. Rice C.M. Dubuisson J. J. Virol. 1997; 71: 697-704Crossref PubMed Google Scholar). This oligomer was thought for a long time to be the prebudding form of the functional complex (12Op De Beeck A. Cocquerel L. Dubuisson J. J. Gen. Virol. 2001; 82: 2589-2595Crossref PubMed Scopus (132) Google Scholar), which is present at the surface of HCV particles (13Op De Beeck A. Voisset C. Bartosch B. Ciczora Y. Cocquerel L. Keck Z. Foung S. Cosset F.L. Dubuisson J. J. Virol. 2004; 78: 2994-3002Crossref PubMed Scopus (190) Google Scholar) and is involved in viral entry. Recent investigation of the E1E2 complex incorporated into HCVcc challenges this notion by proving the existence of large high molecular weight complexes stabilized by disulfide bridges (14Vieyres G. Thomas X. Descamps V. Duverlie G. Patel A.H. Dubuisson J. J. Virol. 2010; 84: 10159-10168Crossref PubMed Scopus (169) Google Scholar). HCV E2 is responsible for virion attachment to target cells and can bind different receptors including several capture molecules, the CD81 tetraspanin, and the scavenger receptor BI (for review, see Refs. 6Bartosch B. Cosset F.L. Virology. 2006; 348: 1-12Crossref PubMed Scopus (135) Google Scholar and 7Cocquerel L. Voisset C. Dubuisson J. J. Gen. Virol. 2006; 87: 1075-1084Crossref PubMed Scopus (161) Google Scholar). Recently, a three-dimensional structural model of E2 has been proposed as a class II fusion protein (15Krey T. d'Alayer J. Kikuti C.M. Saulnier A. Damier-Piolle L. Petitpas I. Johansson D.X. Tawar R.G. Baron B. Robert B. England P. Persson M.A. Martin A. Rey F.A. PLoS Pathog. 2010; 6: e1000762Crossref PubMed Scopus (198) Google Scholar) based on the determination of its disulfide bonds which suggested that it can act alone to mediate binding and membrane fusion. However, both E1 and E2 appear to possess domains implicated in fusion (16Lavillette D. Pécheur E.I. Donot P. Fresquet J. Molle J. Corbau R. Dreux M. Penin F. Cosset F.L. J. Virol. 2007; 81: 8752-8765Crossref PubMed Scopus (149) Google Scholar, 17Russell R.S. Kawaguchi K. Meunier J.C. Takikawa S. Faulk K. Bukh J. Purcell R.H. Emerson S.U. J. Viral. Hepat. 2009; 16: 621-632Crossref PubMed Scopus (28) Google Scholar, 18Rothwangl K.B. Rong L. Virol. J. 2009; 6: 12Crossref PubMed Scopus (8) Google Scholar, 19Owsianka A.M. Timms J.M. Tarr A.W. Brown R.J. Hickling T.P. Szwejk A. Bienkowska-Szewczyk K. Thomson B.J. Patel A.H. Ball J.K. J. Virol. 2006; 80: 8695-8704Crossref PubMed Scopus (216) Google Scholar). Moreover, several antibodies directed against E1 are able to neutralize cell entry, presumably at a stage distinct from receptor binding (20Dreux M. Pietschmann T. Granier C. Voisset C. Ricard-Blum S. Mangeot P.E. Keck Z. Foung S. Vu-Dac N. Dubuisson J. Bartenschlager R. Lavillette D. Cosset F.L. J. Biol. Chem. 2006; 281: 18285-18295Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 21Keck Z.Y. Sung V.M. Perkins S. Rowe J. Paul S. Liang T.J. Lai M.M. Foung S.K. J. Virol. 2004; 78: 7257-7263Crossref PubMed Scopus (93) Google Scholar, 22Pietschmann T. Kaul A. Koutsoudakis G. Shavinskaya A. Kallis S. Steinmann E. Abid K. Negro F. Dreux M. Cosset F.L. Bartenschlager R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7408-7413Crossref PubMed Scopus (615) Google Scholar). Therefore, the role of E1 in HCV infection remains unclear. The two transmembrane domains of the E1E2 heterodimer were shown to be important for different functions and interactions between the two glycoproteins. Studies of mutations occurring in conserved regions and analyses using cross-neutralizing antibodies have shown that these domains are involved in ER retention, heterodimerization of E1E2 on the surface of the viral particles, and even fusion between viral and cellular membranes (23Cocquerel L. Op de Beeck A. Lambot M. Roussel J. Delgrange D. Pillez A. Wychowski C. Penin F. Dubuisson J. EMBO J. 2002; 21: 2893-2902Crossref PubMed Scopus (112) Google Scholar, 24Ciczora Y. Callens N. Montpellier C. Bartosch B. Cosset F.L. Op de Beeck A. Dubuisson J. J. Gen. Virol. 2005; 86: 2793-2798Crossref PubMed Scopus (46) Google Scholar, 25Op De Beeck A. Montserret R. Duvet S. Cocquerel L. Cacan R. Barberot B. Le Maire M. Penin F. Dubuisson J. J. Biol. Chem. 2000; 275: 31428-31437Abstract Full Text Full Text PDF PubMed Google Scholar, 26Ciczora Y. Callens N. Penin F. Pécheur E.I. Dubuisson J. J. Virol. 2007; 81: 2372-2381Crossref PubMed Scopus (73) Google Scholar, 27Patel J. Patel A.H. McLauchlan J. Virology. 2001; 279: 58-68Crossref PubMed Scopus (63) Google Scholar, 28Mottola G. Jourdan N. Castaldo G. Malagolini N. Lahm A. Serafini-Cessi F. Migliaccio G. Bonatti S. J. Biol. Chem. 2000; 275: 24070-24079Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The aim of this study was to characterize interactions between E1 and E2 and the cross-talk between these domains for conformational changes during entry. We assume that such domains of E1 and E2 will have co-evolved inside a given genotype to optimize their interactions and allow efficient entry. In this report we identified non optimal intergenotypic heterodimers that we used to identify less conserved domains involved in E1E2 interactions. We focused on E1E2 intergenotypic heterodimers between H77 (gt1a) and Con1 (gt1b) strains, and we generated chimeras in E1 by substituting H77 for Con1 sequences and vice versa to restore optimal entry function. We discovered that both the ectodomain and transmembrane domain are involved in the cross-talk, taking part during the conformational changes required for entry. Interestingly we show that the N terminus of E1, more precisely the AIL motif, and the transmembrane of E1 H77 need to be homogenous, which is to say from the same strain, to achieve optimal entry. This interaction is crucial for the entry of H77/JFH1 HCVcc chimera and seems to be genotype-dependent, as these interactions are not crucial for Con1. Thus, the specific interactions between E1 and E2 vary between strains. Huh-7 (29Nakabayashi H. Taketa K. Miyano K. Yamane T. Sato J. Cancer Res. 1982; 42: 3858-3863PubMed Google Scholar), Huh7.5 (30Blight K.J. McKeating J.A. Marcotrigiano J. Rice C.M. J. Virol. 2003; 77: 3181-3190Crossref PubMed Scopus (291) Google Scholar), and 293T (ATCC CRL-1573) cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Perbio), 50 IU/ml penicillin, and 50 μg/ml streptomycin (Invitrogen). All chimeric E1E2 heterodimers have been constructed by PCR and/or digestion between genotype 1a strain H77 (31Yanagi M. Purcell R.H. Emerson S.U. Bukh J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 8738-8743Crossref PubMed Scopus (457) Google Scholar) (GenBankTM accession number AF009606) and 1b strain Con1 (32Lohmann V. Körner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2490) Google Scholar) (GenBankTM accession number AJ238799). All mutants were verified by sequencing. For infection assays and Western blots, HCVpp were produced as previously described (3Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (944) Google Scholar) from 293T cells cotransfected with a murine leukemia virus (MLV) Gag-Pol packaging construct, an MLV-based transfer vector encoding the green fluorescent protein, and each of the E1E2 expression constructs. For Western blotting and co-immunoprecipitation assays, the pseudoparticles were purified and concentrated from the cell culture medium by ultracentrifugation at 82,000 × g for 1 h 45 min at 4 °C through 1.5 ml of a 20% sucrose cushion. Viral pellets were suspended in phosphate-buffered saline (PBS) to concentrate the viral particles 100-fold. As a control for infection assays and co-immunoprecipitation assays, pseudoparticles devoid of viral glycoproteins were produced in parallel. Viral pellets were subjected to Western blot analysis using a mouse anti-HCV E1 antibody (IGH204) (Innogenetics), a mouse anti-HCV E2 antibody (H52) (33Flint M. Maidens C. Loomis-Price L.D. Shotton C. Dubuisson J. Monk P. Higginbottom A. Levy S. McKeating J.A. J. Virol. 1999; 73: 6235-6244Crossref PubMed Google Scholar), and a goat anti-MLV-CA antibody (anti-p30; Viromed). Viral pellet samples were mixed with 6× loading buffer (375 mm Tris-HCl, pH 6.8, 3% sodium dodecyl sulfate (SDS), 10% glycerol, and 0.06% bromphenol blue), and the samples were analyzed by electrophoresis in 12% polyacrylamide gels in the presence of 0.1% SDS. After protein transfer onto nitrocellulose filters, the blots were blocked in Tris-buffered saline (1 m, pH 7.4) with 5% milk powder and 0.1% Tween 20 (34Bowen W.D. Walker J.M. de Costa B.R. Wu R. Tolentino P.J. Finn D. Rothman R.B. Rice K.C. J. Pharmacol. Exp. Ther. 1992; 262: 32-40PubMed Google Scholar). The blots were probed with appropriate primary and secondary antibodies (1:10,000-diluted horseradish peroxidase-conjugated anti-mouse or anti-goat; Dako) in Tris-buffered saline, 5% milk, 0.1% Tween 20. Bound enzyme-labeled antibody was visualized using an enhanced chemiluminescence kit (SuperSignal West Pico chemiluminescent substrate; Pierce). To perform immunoprecipitation assays, the pelleted virions were lysed in immunoprecipitation buffer (20 mm Hepes, pH 7.5, 1 mm EGTA, 1 mm EDTA, 150 mm NaCl, and 1% Triton X-100), and the medium containing HCVpp was precleared by overnight incubation with a 1:1 mixture of protein A- and protein G-Sepharose beads (Amersham Biosciences) at 4 °C. After a centrifugation at 13,000 × g at 4 °C for 5 min, the supernatants were incubated with the conformation-dependent anti-E2 monoclonal antibody AR3A (35Law M. Maruyama T. Lewis J. Giang E. Tarr A.W. Stamataki Z. Gastaminza P. Chisari F.V. Jones I.M. Fox R.I. Ball J.K. McKeating J.A. Kneteman N.M. Burton D.R. Nat. Med. 2008; 14: 25-27Crossref PubMed Scopus (489) Google Scholar) for 2 h at 4 °C, and the immune complexes were precipitated using a 1:1 mixture of protein A- and protein G-Sepharose beads for 1h at 4 °C. The complexes were washed three times with immunoprecipitation buffer (50 mm NaCl, 50 mm Tris, pH 7.5, 10 mm EDTA) and analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blot using anti-E1 (IGH204) and anti-E2 (H52) antibodies. Supernatants containing HCVpp were harvested 36 h after transfection and filtered through 0.45-μm-pore-size membranes. Huh-7 target cells (4 × 104 cells/well in 24-well plates) were incubated with different dilutions of HCVpp harboring the chimeric glycoproteins for 4 h at 37 °C. Supernatants were removed, and cells were incubated in complete medium for 72 h at 37 °C. Cells were detached and analyzed by FACS Canto II (BD Biosciences) for GFP expression. E1E2-transfected cells were lysed in pulldown lysis buffer (50 mm Tris HCl, pH 7.4, 150 mm NaCl, 20 mm Imidazole, 2 mm Tris(2-carboxyethyl)phosphine, and 1% Triton X-100). Cell lysates were incubated or not with a recombinant protein containing the large extracellular loop of human CD81 fused to a His6 tag (soluble CD81-LEL-His6) overnight at 4 °C. Then, with nickel-nitrilotriacetic acid magnetic agarose beads and the BioSprint 15 Work station of Qiagen, the lysates were washed with NPI-20-T buffer (50 mm NaH2PO4, 300 mm NaCl, 20 mm imidazole, 0.05% Tween 20, pH 8) and eluted with a NPI-250-T buffer (50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole, 0.05% Tween 20, pH 8). Eluted samples were incubated with non-denaturing buffer and analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blot using anti-E1 (IGH204), anti-E2 (H52), and anti-CD81 (JS81, BD Biosciences) antibodies. 293T “donor” cells (2.5 × 105 cells/well seeded in six-well tissue culture dishes 24 h before transfection) were cotransfected using calcium phosphate reagent with 10 ng of E1E2 chimeric heterodimers and 20 ng of an HIV-1 long terminal repeat (LTR) luciferase reporter plasmid (a kind gift of Françoise Bex, Institut de Recherches Microbiologiques Jean-Marie Wiame) (36Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (119) Google Scholar). As a negative control, cells were cotransfected with 10 ng of empty phCMV plasmid and 20 ng of the HIV-1-LTR-luciferase reporter plasmid. Twelve hours later, transfected cells were detached with Versene (0.53 mm EDTA; Invitrogen) and reseeded at the same concentration (105 cells/well) in 6-well plates. Huh-7-Tat indicator cells (4 × 105 cells/well), detached with EDTA and washed, were then added to the transfected cells. After 24 h of cocultivation, the cells were washed with PBS, incubated for 5 min in a pH 5 fusion buffer (130 mm NaCl, 15 mm sodium citrate, 10 mm MES, 5 mm HEPES), and then washed 3 times with medium. The luciferase activity was measured 24 h later using a luciferase assay kit according to the manufacturer's instructions (Promega). To generate infectious HCV RNAs, pJFH-1=S1a-NS2–1a2a VPL, termed H77/JFH1, and mutant were linearized at the 3′ end by XbaI digestion and were treated with Mung Bean nuclease. Purified linearized DNAs were used as templates for in vitro transcription with the RiboMAXTM (Promega Corp.). In vitro transcribed RNA was used to electroporate Huh7.5 cells using Gene Pulser II apparatus (Bio-Rad) in an L3 laboratory, according to European safety regulations, and cells were cultured under standard conditions. Supernatant infectivity titers were determined as focus-forming units (FFUs) per ml. Huh7.5 cells were infected with different dilutions of culture supernatants. 2 days post-infection, FFUs were visualized after NS5A immunostaining as described previously (37Tellinghuisen T.L. Foss K.L. Treadaway J. PLoS Pathog. 2008; 4: e1000032Crossref PubMed Scopus (333) Google Scholar). FFU calculations were based on counts of NS5A-positive cells. For the kinetic assays, producer cells were infected with a multiplicity of infection of 0.04. For virus spread, Huh7.5 producer cells were split and analyzed by FACS Canto II by NS5A immunostaining. HCV core protein was quantified using the Trak-C Core ELISA (Ortho Clinical Diagnostics, Neckargemünd, Germany) according to the manufacturer's instructions. To characterize the role of less conserved domains of the E1E2 heterodimer involved in cross-talk between either glycoprotein during their conformational changes in entry, we analyzed intergenotypic E1E2 complexes derived from E1 and E2 of different genotypes. The infectivity of HCVpp generated using E1 and E2 expressed in trans from individual plasmids indicated that H77 E1 (gt1a) forms functional heterodimers when associated with E2 derived from all genotypes tested from 1b, 2a, 3, 4, and 5 (Fig. 1A). Conversely, Con1 E1 (gt1b) does not form functional heterodimers when associated with E2 from H77 (gt1a) or JFH1 (gt2a) strains. Our study was focused on combinations using H77 and Con1 strains because of the use of antibodies against E1 (IGH204) and E2 (H52), which recognized linear epitopes on both strains. For a more natural expression context in producer cells, we decided to compare the results of E1E2 intergenotypic heterodimer for H77 and Con1 expressed in cis. These conditions allowed the production of equal amounts of E1 and E2 in HCVpp producer cells. Moreover, the HCVpp titers were increased 10-fold, improving the sensitivity of the assay as has been previously described (3Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (944) Google Scholar, 38Sandrin V. Boulanger P. Penin F. Granier C. Cosset F.L. Bartosch B. J. Gen. Virol. 2005; 86: 3189-3199Crossref PubMed Scopus (46) Google Scholar, 39Bartosch B. Vitelli A. Granier C. Goujon C. Dubuisson J. Pascale S. Scarselli E. Cortese R. Nicosia A. Cosset F.L. J. Biol. Chem. 2003; 278: 41624-41630Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Western blot analysis on producer cells lysates did not indicate any difference in expression level (Fig. 1B). Furthermore, Western blot analysis on purified HCVpp indicated that the level of Con1 E1 incorporated onto HCVpp harboring intergenotypic (heterogeneous) complexes (chimera #2) was similar to the quantity of E1 incorporated onto infectious HCVpp harboring the wild type (homogenous) Con1 complex. Similarly, H77 E2 was incorporated onto HCVpp harboring intergenotypic (heterogeneous) complexes (chimera #2) in a similar quantity to the H77 E2 incorporated onto infectious HCVpp E1E2 wt (homogenous) H77 (Fig. 1B). As with the in trans system, the expression of Con1 E1/H77 E2 in cis (chimera #2) from a polyprotein precursor still led to the production of HCVpp with a decreased titer compared with the wt homogenous combination or the H77 E1/Con1 E2 (chimera #4) mirror combination (Fig. 1D). Correlated with the biochemical analysis, these results indicated that the reduced infectivity observed with the Con1 E1/H77 E2 heterodimer (chimera #2) was not related to alterations of expression and incorporation of E1E2 heterodimers but rather to a cell entry defect (Fig. 1, B and D). Furthermore, compared with the mean 2-log decrease in titer in trans, we obtained a 1-log decrease in cis. This difference may be linked to the fact that the folding of the heterodimer is not optimal when E1 and E2 are expressed in trans as the folding of E1 and E2 are dependent on each other. Therefore, we decided to make subsequent chimera constructions in cis. To further characterize the chimera Con1 E1/H77 E2, we wondered whether its non-optimal infectivity was linked to suboptimal recognition of the CD81 receptor. Using pulldown assays with soluble CD81-LEL harboring a His6 tag, Western blotting analyses indicated that an equal quantity of E1 and E2 was co-immunoprecipitated, indicating that CD81 binding was not impaired in the different constructions. As a control, the input of CD81 precipitated was similar in all cases and E1E2 was not detected without soluble CD81 (Fig. 1C). To determine which domains are involved in the reduction of infection of the intergenotypic Con1 E1/H77 E2 heterodimer (chimera #2), we constructed chimeric E1 by substituting either the ectodomain or transmembrane domain (tmd) of H77 and Con1 strains, respectively. Biochemical analysis indicated that the different chimeric heterodimers had no alterations of expression and incorporation of E1E2 heterodimers (Fig. 2A). To ensure that the glycoproteins incorporated onto the particles were well associated on a conformational heterodimer, we carried out immunoprecipitation of E1E2 heterodimers from purified HCVpp using the AR3A conformational anti-E2 antibody. Western blotting analyses of precipitated complexes indicated that the different constructions displayed well-folded and associated E1E2 heterodimers on particles in similar proportions compared with the wild type heterodimers (Fig. 2B). To investigate further, pulldown assays with soluble CD81-LEL indicated that an equal quantity of E1 and E2 were co-immunoprecipitated, indicating that CD81 binding was not impaired in the different constructions (Fig. 2C). Therefore, the entry properties of chimeric heterodimers are not linked to conformation or assembly problems on the particles. We next investigated the infectivity of the HCVpp harboring the different E1E2 chimeras generated (Fig. 2D). The titers of the different constructs indicated that the ectodomain was responsible for the loss of titer in the Con1 E1/H77 E2 combination (chimera #2). Indeed, compared with the titer of wild type H77, the introduction of the ectodomain led to a 2-log decrease (chimera #3). On the contrary, the substitution of the transmembrane of H77 E1 with the one of Con1 (chimera #1) did not modify the titer compared with the wild type H77. This may suggest that the tmd does not play a role in the function of intergenotypic heterodimer. However, when the titer of the Con1 E1/H77 E2 combination (chimera #2) was compared with the titer of the H77 harboring the sole ectodomain of Con1 E1 (chimera #3), there was a 1-log difference, which in this context did indicate a role for the tmd. Interestingly, when the mirror chimera was generated by introducing the sole ectodomain or tmd of H77 E1 into the wt Con1 E1E2, the conclusion was similar. Indeed, even though the H77 E1/Con1 E2 combination (chimera #4) is optimal for entry, the introduction of either the transmembrane domain (chimera #6) or the ectodomain (chimera #5) alone leads to a 6- and 10-fold reduction in titer, respectively, compared with HCVpp harboring Con1 wt complex. Therefore, depending on the strain origin of E2, the full-length ectodomain and tmd have different roles in heterodimer functionality. As the ectodomain and tmd are important for entry, we first decided to determine which amino acids in the transmembrane domains were responsible for the non-optimal functionality of chimeras #3 and #5 observed in Fig. 2. Based on sequence alignment analysis of the transmembrane domain of H77 and Con1, we found 4-amino acid differences at position 359, 362, 373, and 375 (Fig. 3A). To analyze their respective contribution, we first mutated each residue individually in the chimeric heterodimer Con1 harboring the ectodomain of H77 E1 (chimera #5) leading to the reintroduction of the H77 amino acid in the transmembrane domain. However, the single substitutions L359I, Y362F, I373V, or M375L did not increase the titer of chimera #5 to the level of the fully infectious chimera #4 (Fig. 3B). We then generated different combinations of double and triple substitutions. Infection assays using these HCVpp indicated that three amino acid mutations are necessary to restore titer; L359I, I373V, and M375L (Fig. 3B). The expression and incorporation on HCVpp of the different heterodimers were verified by Western blot and were seen to be at equivalent levels (data not shown). The fact that incorporation is not affected is consistent with previous studies that demonstrated that these amino acids were not implicated in heterodimerization (26Ciczora Y. Callens N. Penin F. Pécheur E.I. Dubuisson J. J. Virol. 2007; 81: 2372-2381Crossref PubMed Scopus (73) Google Scholar). As the transmembrane of Con1 seemed also to have a role in the functionality of chimeric heterodimers, we introduced the opposite substitution in the mirror chimeric heterodimer #3 (Fig. 3C). The H77 construct harboring the ectodomain of Con1 (chimera #3) was only weakly infectious, whereas the H77 E2 when associated with the Con1 E1 (chimera #2) was less affected (Fig. 3C), so we reintroduced Con1 amino acids into the H77 tmd of chimera #3. We obtained comparable results with heterodimers harboring the triple substitution I359L, V373I, and L375M, increasing the titer of chimera #3 (Fig. 3C). Therefore, regardless of the strain, the same three amino acids are important for infectivity of the HCVpp harboring chimeric heterodimers. We next wondered which region of the H77 E1 ectodomain is needed for heterodimer functionality. Based on the sequence alignment of H77 and Con1 E1, we established different boundaries for the construction of chimeras with the 123 (chimera #7) or 36 (chimera #8) N-terminal residues from Con1 in the H77 E1 (Fig. 3A). As for other chimeras, biochemical analysis indicated that expression, incorporation, conformation, and CD81 binding capacity were similar for each construction (Fig. 4, A–C). Infection assays using HCVpp harb" @default.
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• W2142582216 date "2011-07-01" @default.
• W2142582216 modified "2023-10-17" @default.
• W2142582216 title "Identification of Interactions in the E1E2 Heterodimer of Hepatitis C Virus Important for Cell Entry" @default.
• W2142582216 cites W1562770137 @default.
• W2142582216 cites W1825201765 @default.
• W2142582216 cites W1964786592 @default.
• W2142582216 cites W1965513749 @default.
• W2142582216 cites W1967491261 @default.
• W2142582216 cites W1970324388 @default.
• W2142582216 cites W1976598372 @default.
• W2142582216 cites W1976844675 @default.
• W2142582216 cites W1982315879 @default.
• W2142582216 cites W2003579943 @default.
• W2142582216 cites W2004270276 @default.
• W2142582216 cites W2008474303 @default.
• W2142582216 cites W2012420380 @default.
• W2142582216 cites W2023645777 @default.
• W2142582216 cites W2030371794 @default.
• W2142582216 cites W2032955836 @default.
• W2142582216 cites W2038454674 @default.
• W2142582216 cites W2042994480 @default.
• W2142582216 cites W2044193462 @default.
• W2142582216 cites W2060483294 @default.
• W2142582216 cites W2076883913 @default.
• W2142582216 cites W2083579867 @default.
• W2142582216 cites W2092637586 @default.
• W2142582216 cites W2106399441 @default.
• W2142582216 cites W2108463852 @default.
• W2142582216 cites W2110842901 @default.
• W2142582216 cites W2114132885 @default.
• W2142582216 cites W2116192835 @default.
• W2142582216 cites W2116753660 @default.
• W2142582216 cites W2118273588 @default.
• W2142582216 cites W2123693893 @default.
• W2142582216 cites W2125853173 @default.
• W2142582216 cites W2129729109 @default.
• W2142582216 cites W2130661443 @default.
• W2142582216 cites W2135495604 @default.
• W2142582216 cites W2140799443 @default.
• W2142582216 cites W2141398361 @default.
• W2142582216 cites W2143312809 @default.
• W2142582216 cites W2145943989 @default.
• W2142582216 cites W2147126871 @default.
• W2142582216 cites W2150010918 @default.
• W2142582216 cites W2151898297 @default.
• W2142582216 cites W2154115492 @default.
• W2142582216 cites W2154898842 @default.
• W2142582216 cites W2155798686 @default.
• W2142582216 cites W2157712520 @default.
• W2142582216 cites W2158819883 @default.
• W2142582216 cites W2164508705 @default.
• W2142582216 cites W2167714763 @default.
• W2142582216 doi "https://doi.org/10.1074/jbc.m110.213942" @default.
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