Cholesterol sensing by CD81 is important for hepatitis C virus entry
2020; Elsevier BV; Volume: 295; Issue: 50 Linguagem: Inglês
10.1074/jbc.ra120.014761
ISSN1083-351X
AutoresMachaela Palor, Lenka Stejskal, Piya Mandal, Annasara Lenman, María Pía Alberione, Jared Kirui, Rebecca Moeller, Stefan Ebner, Felix Meissner, Gisa Gerold, Adrian J. Shepherd, Joe Grove,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoCD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease. CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease. Binding of the E2 glycoprotein of hepatitis C virus (HCV) to the large extracellular loop of CD81 is a defining event in the entry of HCV (1Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Binding of hepatitis C virus to CD81.Science. 1998; 282 (9794763): 938-94110.1126/science.282.5390.938Crossref PubMed Scopus (1766) Google Scholar) and is targeted by multiple broadly neutralizing antibodies, thus placing this molecular interaction at the forefront of current HCV vaccine development (2Kinchen V.J. Zahid M.N. Flyak A.I. Soliman M.G. Learn G.H. Wang S. Davidson E. Doranz B.J. Ray S.C. Cox A.L. Crowe Jr., J.E. Bjorkman P.J. Shaw G.M. Bailey J.R. Broadly neutralizing antibody mediated clearance of human hepatitis C virus infection.Cell Host Microbe. 2018; 24 (30439341): 717-730.e510.1016/j.chom.2018.10.012Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 3Flyak A.I. Ruiz S. Colbert M.D. Luong T. Crowe Jr., J.E. Bailey J.R. Bjorkman P.J. HCV broadly neutralizing antibodies use a CDRH3 disulfide motif to recognize an E2 glycoprotein site that can be targeted for vaccine design.Cell Host Microbe. 2018; 24 (30439340): 703-716.e310.1016/j.chom.2018.10.009Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Although the importance of CD81 in HCV entry is well-established, the precise details of E2–CD81 interaction have yet to be defined, and the molecular determinants of CD81 receptor activity are only partially understood (4Banse P. Moeller R. Bruening J. Lasswitz L. Kahl S. Khan A.G. Marcotrigiano J. Pietschmann T. Gerold G. CD81 receptor regions outside the large extracellular loop determine hepatitis C virus entry into hepatoma cells.Viruses. 2018; 10 (29677132): 20710.3390/v10040207Crossref Scopus (7) Google Scholar). CD81 is a prototypical member of the tetraspanin superfamily. Tetraspanins are small integral membrane proteins defined by their four transmembrane domains separated by intra-/extracellular loops. Highly conserved cysteine residues stabilize tetraspanin tertiary structure through disulfide bridges and provide sites for post-translational palmitoylation, which influences tetraspanin membrane segregation (5Charrin S. Jouannet S. Boucheix C. Rubinstein E. Tetraspanins at a glance.J. Cell Sci. 2014; 127 (25128561): 3641-364810.1242/jcs.154906Crossref PubMed Scopus (254) Google Scholar, 6Charrin S. Le Naour F. Silvie O. Milhiet P.-E. Boucheix C. Rubinstein E. Lateral organization of membrane proteins: tetraspanins spin their web.Biochem. J. 2009; 420 (19426143): 133-15410.1042/BJ20082422Crossref PubMed Scopus (312) Google Scholar). Largely without cognate ligands, tetraspanins participate indirectly in a wide variety of cell-biological processes through their interactions with partner proteins, which they organize into functional complexes (7Gerold G. Meissner F. Bruening J. Welsch K. Perin P.M. Baumert T.F. Vondran F.W. Kaderali L. Marcotrigiano J. Khan A.G. Mann M. Rice C.M. Pietschmann T. Quantitative proteomics identifies serum response factor binding protein 1 as a host factor for hepatitis C virus entry.Cell Rep. 2015; 12 (26212323): 864-87810.1016/j.celrep.2015.06.063Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 8Bruening J. Lasswitz L. Banse P. Kahl S. Marinach C. Vondran F.W. Kaderali L. Silvie O. Pietschmann T. Meissner F. Gerold G. Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB.PLoS Pathog. 2018; 14 (30024968)e100711110.1371/journal.ppat.1007111Crossref PubMed Scopus (27) Google Scholar). For example, CD81 facilitates the assembly of the B-cell receptor complex and is therefore essential for normal antibody responses. CD81 performs this role via partnership with CD19; first by chaperoning CD19 through the secretory pathway and then by dictating its cell-surface distribution, permitting proper assembly of the B-cell receptor complex upon activation (9van Zelm M.C. Smet J. Adams B. Mascart F. Schandené L. Janssen F. Ferster A. Kuo C.-C. Levy S. van Dongen J.J.M. van der Burg M. CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency.J. Clin. Invest. 2010; 120 (20237408): 1265-127410.1172/JCI39748Crossref PubMed Scopus (281) Google Scholar, 10Cherukuri A. Shoham T. Sohn H.W. Levy S. Brooks S. Carter R. Pierce S.K. The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21–B cell antigen receptor complexes into signaling-active lipid rafts.J. Immunol. 2004; 172 (14688345): 370-38010.4049/jimmunol.172.1.370Crossref PubMed Scopus (113) Google Scholar, 11Shoham T. Rajapaksa R. Boucheix C. Rubinstein E. Poe J.C. Tedder T.F. Levy S. The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment.J. Immunol. 2003; 171 (14530327): 4062-407210.4049/jimmunol.171.8.4062Crossref PubMed Scopus (91) Google Scholar, 12Mattila P.K. Feest C. Depoil D. Treanor B. Montaner B. Otipoby K.L. Carter R. Justement L.B. Bruckbauer A. Batista F.D. The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling.Immunity. 2013; 38 (23499492): 461-47410.1016/j.immuni.2012.11.019Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Through other molecular partnerships, CD81 has been implicated in additional physiological processes such as T-cell receptor signaling, cell migration, growth factor signaling, sperm–egg fusion, and most recently, biological aging, potentially through its interaction with TMEM2 (13Rocha-Perugini V. Zamai M. González-Granado J.M. Barreiro O. Tejera E. Yañez-Mó M. Caiolfa V.R. Sanchez-Madrid F. CD81 controls sustained T cell activation signaling and defines the maturation stages of cognate immunological synapses.Mol. Cell Biol. 2013; 33 (23858057): 3644-365810.1128/MCB.00302-13Crossref PubMed Scopus (41) Google Scholar, 14Brimacombe C.L. Wilson G.K. Hübscher S.G. McKeating J.A. Farquhar M.J. A role for CD81 and hepatitis C virus in hepatoma mobility.Viruses. 2014; 6 (24662676): 1454-147210.3390/v6031454Crossref PubMed Scopus (8) Google Scholar, 15Diao J. Pantua H. Ngu H. Komuves L. Diehl L. Schaefer G. Kapadia S.B. Hepatitis C virus induces epidermal growth factor receptor activation via CD81 binding for viral internalization and entry.J. Virol. 2012; 86 (22855500): 10935-1094910.1128/JVI.00750-12Crossref PubMed Scopus (93) Google Scholar, 16Rubinstein E. Ziyyat A. Prenant M. Wrobel E. Wolf J.-P. Levy S. Le Naour F. Boucheix C. Reduced fertility of female mice lacking CD81.Dev. Biol. 2006; 290 (16380109): 351-35810.1016/j.ydbio.2005.11.031Crossref PubMed Scopus (155) Google Scholar, 17Ohnami N. Nakamura A. Miyado M. Sato M. Kawano N. Yoshida K. Harada Y. Takezawa Y. Kanai S. Ono C. Takahashi Y. Kimura K. Shida T. Miyado K. Umezawa A. CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte.Biol. Open. 2012; 1 (23213457): 640-64710.1242/bio.20121420Crossref PubMed Scopus (40) Google Scholar, 18Jin Y. Takeda Y. Kondo Y. Tripathi L.P. Kang S. Takeshita H. Kuhara H. Maeda Y. Higashiguchi M. Miyake K. Morimura O. Koba T. Hayama Y. Koyama S. Nakanishi K. et al.Double deletion of tetraspanins CD9 and CD81 in mice leads to a syndrome resembling accelerated aging.Sci. Rep. 2018; 8 (29572511)514510.1038/s41598-018-23338-xCrossref PubMed Scopus (23) Google Scholar, 19Schinzel R.T. Higuchi-Sanabria R. Shalem O. Moehle E.A. Webster B.M. Joe L. Bar-Ziv R. Frankino P.A. Durieux J. Pender C. Kelet N. Kumar S.S. Savalia N. Chi H. Simic M. et al.The hyaluronidase, TMEM2, promotes ER homeostasis and longevity independent of the UPRER.Cell. 2019; 179 (31761535): 1306-1318.e1810.1016/j.cell.2019.10.018Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Aside from these physiological functions, CD81 is also commandeered by diverse infectious pathogens. It participates in the cell-surface assembly of both HIV and influenza A virus, a function that may be linked to the apparent affinity of CD81 for membrane structures with high curvature (20Nydegger S. Khurana S. Krementsov D.N. Foti M. Thali M. Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1.J. Cell Biol. 2006; 173 (16735575): 795-80710.1083/jcb.200508165Crossref PubMed Scopus (187) Google Scholar, 21He J. Sun E. Bujny M.V. Kim D. Davidson M.W. Zhuang X. Dual function of CD81 in influenza virus uncoating and budding.PLoS Pathog. 2013; 9 (24130495)e100370110.1371/journal.ppat.1003701Crossref PubMed Scopus (67) Google Scholar, 22Shaw M.L. Stone K.L. Colangelo C.M. Gulcicek E.E. Palese P. Cellular proteins in influenza virus particles.PLoS Pathog. 2008; 4 (18535660)e100008510.1371/journal.ppat.1000085Crossref PubMed Scopus (247) Google Scholar, 23Grove J. Super-resolution microscopy: a virus' eye view of the cell.Viruses. 2014; 6 (24651030): 1365-137810.3390/v6031365Crossref PubMed Scopus (27) Google Scholar). CD81 also negatively regulates SAMHD1 function, resulting in increased intracellular pools of dNTPs, which in turn favors HIV reverse transcription (24Rocha-Perugini V. Suárez H. Álvarez S. López-Martín S. Lenzi G.M. Vences-Catalán F. Levy S. Kim B. Muñoz-Fernández M.A. Sánchez-Madrid F. Yáñez-Mó M. CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels.Nat. Microbiol. 2017; 2 (28871089): 1513-152210.1038/s41564-017-0019-0Crossref PubMed Scopus (19) Google Scholar). Finally, CD81 is critical for the entry of HCV and Plasmodium sporozoites into human hepatocytes (1Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Binding of hepatitis C virus to CD81.Science. 1998; 282 (9794763): 938-94110.1126/science.282.5390.938Crossref PubMed Scopus (1766) Google Scholar, 25Silvie O. Rubinstein E. Franetich J.-F. Prenant M. Belnoue E. Rénia L. Hannoun L. Eling W. Levy S. Boucheix C. Mazier D. Hepatocyte CD81 is required for Plasmodium falciparumPlasmodium yoelii sporozoite infectivity.Nat. Med. 2003; 9 (12483205): 93-9610.1038/nm808Crossref PubMed Scopus (248) Google Scholar). In summary, CD81 performs molecular scaffolding function in a variety of pathways; a greater understanding of its molecular characteristics will provide novel insights into both physiological and pathological processes. The recent crystal structure of CD81, the first of any tetraspanin, has provided a novel perspective on its molecular biology (26Zimmerman B. Kelly B. McMillan B.J. Seegar T.C.M. Dror R.O. Kruse A.C. Blacklow S.C. Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket.Cell. 2016; 167 (27881302): 1041-1051.e1110.1016/j.cell.2016.09.056Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). CD81's four helical transmembrane domains are arranged in a loose bundle forming an inverted conical shape. Curiously, the transmembrane domains enclose a central intramembrane cavity filled by a single molecule of cholesterol, which is coordinated by hydrogen bonding to the side chains of inward-facing amino acids. Although this observation may have arisen because of the presence of cholesterol in the crystallization buffer, Zimmerman et al. (26Zimmerman B. Kelly B. McMillan B.J. Seegar T.C.M. Dror R.O. Kruse A.C. Blacklow S.C. Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket.Cell. 2016; 167 (27881302): 1041-1051.e1110.1016/j.cell.2016.09.056Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) use biochemical experiments to demonstrate physical association of CD81 with cholesterol. Moreover, this finding is consistent with other reports linking cholesterol to tetraspanin biology (27Charrin S. Manié S. Thiele C. Billard M. Gerlier D. Boucheix C. Rubinstein E. A physical and functional link between cholesterol and tetraspanins.Eur. J. Immunol. 2003; 33 (12938224): 2479-248910.1002/eji.200323884Crossref PubMed Scopus (174) Google Scholar, 28Silvie O. Charrin S. Billard M. Franetich J.-F. Clark K.L. van Gemert G.-J. Sauerwein R.W. Dautry F. Boucheix C. Mazier D. Rubinstein E. Cholesterol contributes to the organization of tetraspanin-enriched microdomains and to CD81-dependent infection by malaria sporozoites.J. Cell Sci. 2006; 119 (16687736): 1992-200210.1242/jcs.02911Crossref PubMed Scopus (95) Google Scholar). Whereas the minor extracellular domain (EC1) was not resolved in the crystal structure (Protein Data Bank code 5TCX (26Zimmerman B. Kelly B. McMillan B.J. Seegar T.C.M. Dror R.O. Kruse A.C. Blacklow S.C. Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket.Cell. 2016; 167 (27881302): 1041-1051.e1110.1016/j.cell.2016.09.056Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar)), CD81's major extracellular domain (EC2) was found to be roughly parallel to the plane of the plasma membrane, analogous to a lid sitting on top of the bundle of transmembrane domains (Fig. 1A and Fig. S2A). Overall, CD81 adopts a compact structure that is likely to project only a few nanometers from the cell surface. However, using molecular dynamic simulations, Zimmerman et al. (26Zimmerman B. Kelly B. McMillan B.J. Seegar T.C.M. Dror R.O. Kruse A.C. Blacklow S.C. Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket.Cell. 2016; 167 (27881302): 1041-1051.e1110.1016/j.cell.2016.09.056Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) demonstrated that the EC2 of CD81 has a propensity to flip up into an extended open conformation (Fig. S2A). Furthermore, removal of cholesterol from the intramembrane cavity during the simulations increased the frequency of conformational switching, suggesting an allosteric link between cholesterol binding and CD81 conformation. These observations indicate that CD81 may have an as-yet unappreciated function as a cholesterol sensor; this feature is likely to be important for its role in scaffolding events occurring at cellular membranes. Although the precise molecular interaction of HCV E2 with the EC2 of CD81 has yet to be structurally defined, the relevant protein domains have been identified (29Drummer H.E. Boo I. Maerz A.L. Poumbourios P. A conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr motif in hepatitis C virus glycoprotein E2 is a determinant of CD81 binding and viral entry.J. Virol. 2006; 80 (16873241): 7844-785310.1128/JVI.00029-06Crossref PubMed Scopus (115) Google Scholar, 30Drummer H.E. Wilson K.A. Poumbourios P. Determinants of CD81 dimerization and interaction with hepatitis C virus glycoprotein E2.Biochem. Biophys. Res. Commun. 2005; 328 (15670777): 251-25710.1016/j.bbrc.2004.12.160Crossref PubMed Scopus (39) Google Scholar, 31Drummer H.E. Wilson K.A. Poumbourios P. Identification of the hepatitis C virus E2 glycoprotein binding site on the large extracellular loop of CD81.J. Virol. 2002; 76 (12368358): 11143-1114710.1128/jvi.76.21.11143-11147.2002Crossref PubMed Scopus (108) Google Scholar, 32Keck Z.-Y. Saha A. Xia J. Wang Y. Lau P. Krey T. Rey F.A. Foung S.K.H. Mapping a region of hepatitis C virus E2 that is responsible for escape from neutralizing antibodies and a core CD81-binding region that does not tolerate neutralization escape mutations.J. Virol. 2011; 85 (21813602): 10451-1046310.1128/JVI.05259-11Crossref PubMed Scopus (84) Google Scholar, 33Kong L. Giang E. Nieusma T. Kadam R.U. Cogburn K.E. Hua Y. Dai X. Stanfield R.L. Burton D.R. Ward A.B. Wilson I.A. Law M. Hepatitis C virus E2 envelope glycoprotein core structure.Science. 2013; 342 (24288331): 1090-109410.1126/science.1243876Crossref PubMed Scopus (294) Google Scholar, 34Higginbottom A. Quinn E.R. Kuo C. Flint M. Wilson L.H. Bianchi E. Nicosia A. Monk P.N. McKeating J.A. Levy S. Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2.J. Virol. 2000; 74 (10729140): 3642-364910.1128/jvi.74.8.3642-3649.2000Crossref PubMed Scopus (191) Google Scholar). The CD81-binding site of HCV E2 comprises discontinuous protein regions, brought together in the 3D structure of the glycoprotein; these regions interact with helices D and E of CD81's EC2, which are presented at the apex of CD81's closed compact structure. Antibodies that prevent this interaction block HCV entry, and cells without CD81 are completely resistant to infection (35Grove J. Hu K. Farquhar M.J. Goodall M. Walker L. Jamshad M. Drummer H.E. Bill R.M. Balfe P. McKeating J.A. A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81.Wellcome Open Res. 2017; 2 (29090272): 8210.12688/wellcomeopenres.12058.1Crossref PubMed Scopus (9) Google Scholar, 36Lindenbach 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. Complete replication of hepatitis C virus in cell culture.Science. 2005; 309 (15947137): 623-62610.1126/science.1114016Crossref PubMed Scopus (1921) Google Scholar, 37Yamamoto S. Fukuhara T. Ono C. Uemura K. Kawachi Y. Shiokawa M. Mori H. Wada M. Shima R. Okamoto T. Hiraga N. Suzuki R. Chayama K. Wakita T. Matsuura Y. Lipoprotein receptors redundantly participate in entry of hepatitis C virus.PLoS Pathog. 2016; 12 (27152966)e100561010.1371/journal.ppat.1005610Crossref PubMed Scopus (51) Google Scholar, 38Flint M. von Hahn T. Zhang J. Farquhar M. Jones C.T. Balfe P. Rice C.M. McKeating J.A. Diverse CD81 proteins support hepatitis C virus infection.J. Virol. 2006; 80 (16943299): 11331-1134210.1128/JVI.00104-06Crossref PubMed Scopus (128) Google Scholar, 39Fofana I. Xiao F. Thumann C. Turek M. Zona L. Tawar R.G. Grunert F. Thompson J. Zeisel M.B. Baumert T.F. A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission.PLoS One. 2013; 8 (23704981)e6422110.1371/journal.pone.0064221Crossref PubMed Scopus (46) Google Scholar, 40Koutsoudakis G. Herrmann E. Kallis S. Bartenschlager R. Pietschmann T. The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells.J. Virol. 2007; 81 (17079281): 588-59810.1128/JVI.01534-06Crossref PubMed Scopus (181) Google Scholar, 41Zhang J. Randall G. Higginbottom A. Monk P. Rice C.M. McKeating J.A. CD81 is required for hepatitis C virus glycoprotein-mediated viral infection.J. Virol. 2004; 78 (14722300): 1448-145510.1128/jvi.78.3.1448-1455.2004Crossref PubMed Scopus (304) Google Scholar, 42Owsianka A. Tarr A.W. Juttla V.S. Lavillette D. Bartosch B. Cosset F.-L. Ball J.K. Patel A.H. Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein.J. Virol. 2005; 79 (16103160): 11095-1110410.1128/JVI.79.17.11095-11104.2005Crossref PubMed Scopus (239) Google Scholar, 43Sabo M.C. Luca V.C. Prentoe J. Hopcraft S.E. Blight K.J. Yi M. Lemon S.M. Ball J.K. Bukh J. Evans M.J. Fremont D.H. Diamond M.S. Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step.J. Virol. 2011; 85 (21543495): 7005-701910.1128/JVI.00586-11Crossref PubMed Scopus (105) Google Scholar, 44Giang E. Dorner M. Prentoe J.C. Dreux M. Evans M.J. Bukh J. Rice C.M. Ploss A. Burton D.R. Law M. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22492964): 6205-621010.1073/pnas.1114927109Crossref PubMed Scopus (254) Google Scholar). The ability of CD81 to recruit molecular partners is also likely to be important for HCV infection; indeed, other HCV entry factors constitutively associate with CD81 (8Bruening J. Lasswitz L. Banse P. Kahl S. Marinach C. Vondran F.W. Kaderali L. Silvie O. Pietschmann T. Meissner F. Gerold G. Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB.PLoS Pathog. 2018; 14 (30024968)e100711110.1371/journal.ppat.1007111Crossref PubMed Scopus (27) Google Scholar, 45Harris H.J. Davis C. Mullins J.G.L. Hu K. Goodall M. Farquhar M.J. Mee C.J. McCaffrey K. Young S. Drummer H. Balfe P. McKeating J.A. Claudin association with CD81 defines hepatitis C virus entry.J. Biol. Chem. 2010; 285 (20375010): 21092-2110210.1074/jbc.M110.104836Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Significantly, HCV entry also seems to be closely linked to cell-surface cholesterol transport: three cholesterol-transporting proteins (SR-B1, LDLR, and NPC1L1) have been implicated in the process (46Ding Q. von Schaewen M. Ploss A. The impact of hepatitis C virus entry on viral tropism.Cell Host Microbe. 2014; 16 (25525789): 562-56810.1016/j.chom.2014.10.009Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Notably, the cholesterol transporter scavenger receptor B-1 (SR-B1) naturally associates with CD81 and also modulates the CD81-dependent invasion of Plasmodium sporozoites into hepatocytes (8Bruening J. Lasswitz L. Banse P. Kahl S. Marinach C. Vondran F.W. Kaderali L. Silvie O. Pietschmann T. Meissner F. Gerold G. Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB.PLoS Pathog. 2018; 14 (30024968)e100711110.1371/journal.ppat.1007111Crossref PubMed Scopus (27) Google Scholar, 47Yalaoui S. Huby T. Franetich J.-F. Gego A. Rametti A. Moreau M. Collet X. Siau A. van Gemert G.-J. Sauerwein R.W. Luty A.J.F. Vaillant J.-C. Hannoun L. Chapman J. Mazier D. et al.Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection.Cell Host Microbe. 2008; 4 (18779054): 283-29210.1016/j.chom.2008.07.013Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 48Rodrigues C.D. Hannus M. Prudêncio M. Martin C. Gonçalves L.A. Portugal S. Epiphanio S. Akinc A. Hadwiger P. Jahn-Hofmann K. Röhl I. van Gemert G.-J. Franetich J.-F. Luty A.J.F. Sauerwein R. et al.Host scavenger receptor SR-BI plays a dual role in the establishment of malaria parasite liver infection.Cell Host Microbe. 2008; 4 (18779053): 271-28210.1016/j.chom.2008.07.012Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The biology of both CD81 and HCV converge on plasma membrane cholesterol; therefore, we set out to investigate how CD81's interaction with cholesterol impacts HCV infection. First, we mutated residues within the cholesterol-binding pocket of CD81. Although all of the tested mutations reduced CD81–cholesterol association, they had varying effects on HCV, both decreasing and increasing virus entry. This suggests the cholesterol-binding pocket of CD81 is important for HCV infection, but viral entry may not be directly dependent on cholesterol association. We performed multiple independent molecular dynamics (MD) simulations of CD81 behavior with and without cholesterol. In support of the report by Zimmerman et al. (26Zimmerman B. Kelly B. McMillan B.J. Seegar T.C.M. Dror R.O. Kruse A.C. Blacklow S.C. Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket.Cell. 2016; 167 (27881302): 1041-1051.e1110.1016/j.cell.2016.09.056Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), we demonstrate a cholesterol-dependent conformational switch of CD81; this is consistent with the notion of cholesterol sensing by CD81. These experiments identified a potential hinging between CD81's EC2 and transmembrane domains. We designed mutations to alter this motion and, therefore, disrupt CD81's cholesterol-sensing mechanism. Mutations that are predicted to confer the open conformation (i.e. the cholesterol-unbound state) reduced HCV entry, whereas mutations that confer the closed conformation (i.e. cholesterol-bound state) enhanced HCV entry. Further characterization of these mutants demonstrate that they exhibit normal cell-surface expression and distribution and retain the ability to chaperone CD19 to the cell surface. However, the open mutant of CD81 exhibits reduced interaction with HCV E2. We also use diverse cell culture–proficient HCV to demonstrate that cholesterol-binding and open conformation mutants of CD81 do not support authentic viral replication. This study provides further insight into the molecular mechanism of cholesterol sensing by CD81 and demonstrates that this activity is important for HCV infection. The crystal structure of CD81 (Protein Data Bank code 5TCX (26Zimmerman B. Kelly B. McMillan B.J. Seegar T.C.M. Dror R.O. Kruse A.C. Blacklow S.C. Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket.Cell. 2016; 167 (27881302): 1041-1051.e1110.1016/j.cell.2016.09.056Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar)) reveals an intramembrane cavity bounded by the four transmembrane domains; this contains a single molecule of cholesterol, which is coordinated by hydrogen bonding to the side chains of two residues, Asn18 and Glu219 (Fig. 1A). We designed a series of mutants to disrupt this interaction: E219A and E219Q, which were previously demonstrated to reduce CD81–cholesterol association (26Zimmerman B. Kelly B. McMillan B.J. Seegar T.C.M. Dror R.O. Kruse A.C. Blacklow S.C. Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket.Cell. 2016; 167 (27881302): 1041-1051.e1110.1016/j.cell.2016.09.056Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), and an N18A/E219A double mutation, which should remove all possibility of hydrogen bonding to cholesterol. Many of the inward-facing residues of CD81's intramembrane cavity have small side chains (e.g. alanine, valine, glycine), this creates a binding pocket to accommodate cholesterol. Therefore, we also mutated four inward-facing residues to tryptophan (V68W/M72W/A108W/V212W), the side chain of which includes a bulky indole group. Structural modeling predicts that these tryptophan residues will fill the cholesterol-binding pocket while maintaining the hydrophobic nature of the transmembrane domains (Fig. 1B). We introduced each of the cholesterol-binding-pocket mutants into Huh-7 CD81 KO cells by lentiviral transduction and confirmed tha
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