Artigo Acesso aberto Revisado por pares

Functional Evolution of the HIV-1 Envelope Glycoprotein 120 Association Site of Glycoprotein 41

2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês

10.1074/jbc.m305223200

ISSN

1083-351X

Autores

Pantelis Poumbourios, Anne L. Maerz, Heidi E. Drummer,

Tópico(s)

Mosquito-borne diseases and control

Resumo

Protein-protein interaction surfaces can exhibit structural plasticity, a mechanism whereby an interface adapts to mutations as binding partners coevolve. The HIV-1 envelope glycoprotein gp120-gp41 complex, which is responsible for receptor attachment and membrane fusion, represents an extreme example of a coevolving complex as up to 35% amino acid sequence divergence has been observed in these proteins among HIV-1 isolates. In this study, the function of conserved gp120 contact residues, Leu593, Trp596, Gly597, Lys601, and Trp610 within the disulfide-bonded region of gp41, was examined in envelope glycoproteins derived from diverse HIV-1 isolates. We found that the gp120-gp41 association function of the disulfide-bonded region is conserved. However, the contribution of individual residues to gp41 folding and/or stability, gp120-gp41 association, membrane fusion function, and viral entry varied from isolate to isolate. In gp120-gp41 derived from the dual-tropic isolate, HIV-189.6, the importance of Trp596 for fusion function was dependent on the chemokine receptor utilized as a fusion cofactor. Thus, the engagement of alternative chemokine receptors may evoke distinct fusion-activation signals involving the site of gp120-gp41 association. An examination of chimeric glycoproteins revealed that the isolate-specific functional contributions of particular gp120-contact residues are influenced by the sequence of gp120 hypervariable regions 1, 2, and 3. These data indicate that the gp120-gp41 association site is structurally and functionally adaptable, perhaps to maintain a functional glycoprotein complex in a setting of host selective pressures driving the rapid coevolution of gp120 and gp41. Protein-protein interaction surfaces can exhibit structural plasticity, a mechanism whereby an interface adapts to mutations as binding partners coevolve. The HIV-1 envelope glycoprotein gp120-gp41 complex, which is responsible for receptor attachment and membrane fusion, represents an extreme example of a coevolving complex as up to 35% amino acid sequence divergence has been observed in these proteins among HIV-1 isolates. In this study, the function of conserved gp120 contact residues, Leu593, Trp596, Gly597, Lys601, and Trp610 within the disulfide-bonded region of gp41, was examined in envelope glycoproteins derived from diverse HIV-1 isolates. We found that the gp120-gp41 association function of the disulfide-bonded region is conserved. However, the contribution of individual residues to gp41 folding and/or stability, gp120-gp41 association, membrane fusion function, and viral entry varied from isolate to isolate. In gp120-gp41 derived from the dual-tropic isolate, HIV-189.6, the importance of Trp596 for fusion function was dependent on the chemokine receptor utilized as a fusion cofactor. Thus, the engagement of alternative chemokine receptors may evoke distinct fusion-activation signals involving the site of gp120-gp41 association. An examination of chimeric glycoproteins revealed that the isolate-specific functional contributions of particular gp120-contact residues are influenced by the sequence of gp120 hypervariable regions 1, 2, and 3. These data indicate that the gp120-gp41 association site is structurally and functionally adaptable, perhaps to maintain a functional glycoprotein complex in a setting of host selective pressures driving the rapid coevolution of gp120 and gp41. The HIV-1 1The abbreviations used are: HIV, human immunodeficiency virus; Env, envelope glycoprotein; V1 to V5, hypervariable regions 1 to 5 of glycoprotein 120; C1 to C5, conserved regions 1 to 5 of gp120; mAb, monoclonal antibody; R5, X4, and R5X4, specificity of Env for the chemokine receptors CCR5, CXCR4, or both CCR5 and CXCR4, respectively; PBS, phosphate-buffered saline; IgG#14, IgG purified from HIV-1-positive human plasma; TCLA, T cell-line adapted; sCD4, soluble CD4; gp, glycoprotein; BHK, baby hamster kidney.1The abbreviations used are: HIV, human immunodeficiency virus; Env, envelope glycoprotein; V1 to V5, hypervariable regions 1 to 5 of glycoprotein 120; C1 to C5, conserved regions 1 to 5 of gp120; mAb, monoclonal antibody; R5, X4, and R5X4, specificity of Env for the chemokine receptors CCR5, CXCR4, or both CCR5 and CXCR4, respectively; PBS, phosphate-buffered saline; IgG#14, IgG purified from HIV-1-positive human plasma; TCLA, T cell-line adapted; sCD4, soluble CD4; gp, glycoprotein; BHK, baby hamster kidney. envelope glycoprotein (Env) complex comprises a trimer of surface-exposed gp120 subunits that are noncovalently associated with a trimer of gp41 transmembrane subunits (1Center R.J. 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Current binding models propose that attachment of CD4 to gp120 leads to a re-organization of the V1V2 and V3 loops and the formation of the chemokine receptor binding site (see Ref. 25Kwong P.D. Wyatt R. Robinson J. Sweet R.W. Sodroski J. Hendrickson W.A. Nature. 1998; 393: 648-659Crossref PubMed Scopus (2502) Google Scholar). The chemokine receptor specificity of an HIV-1 isolate determines its cellular preference or tropism. HIV-1 strains that utilize CCR5 ("R5" strains) preferentially infect CD4+RO+ memory T cells. However, in ∼50% of infected individuals, the onset of symptomatic disease is preceded by the emergence of strains able to utilize CXCR4 ("X4" and "R5X4" dual tropic strains) that broadens viral tropism to both CD4+RO+ memory and CD4+RA+ naive T cells (33Blaak H. van't Wout A.B. Brouwer M. Hooibrink B. Hovenkamp E. Schuitemaker H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1269-1274Crossref PubMed Scopus (227) Google Scholar, 34van Rij R.P. Blaak H. Visser J.A. Brouwer M. Rientsma R. Broersen S. de Roda Husman A.M. Schuitemaker H. J. Clin. Invest. 2000; 106: 1039-1052Crossref PubMed Scopus (96) Google Scholar). The chemokine receptor preference of HIV-1 isolates is determined largely by V3 sequences, with the CCR5 to CXCR4 switch being generally associated with increased positive charge in V3, often in conjunction with sequence changes in V1V2 and C4 (see Ref. 35Hoffman N.G. Seillier-Moiseiwitsch F. Ahn J. Walker J.M. Swanstrom R. J. Virol. 2002; 76: 3852-3864Crossref PubMed Scopus (149) Google Scholar).The fusion activation of gp41 is considered to involve the propagation of a conformational signal from CD4/chemokine receptor-bound gp120 through the sites of gp120-gp41 contact, culminating in the insertion of the fusion peptide into the target membrane and the refolding of the gp41 ectodomain into a six-helix bundle, promoting membrane fusion (15Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar, 19Melikyan G.B. Markosyan R.M. Hemmati H. Delmedico M.K. Lambert D.M. Cohen F.S. J. Cell Biol. 2000; 151: 413-423Crossref PubMed Scopus (478) Google Scholar, 22Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (517) Google Scholar, 23Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1457) Google Scholar). The sites of gp120-gp41 contact have been mapped by in vitro mutagenesis to conserved residues within the disulfide-bonded region of gp41 and in the extremities of C1 and C5 of gp120 (Fig. 1A) (36Helseth E. Olshevsky U. Furman C. Sodroski J. J. Virol. 1991; 65: 2119-2123Crossref PubMed Google Scholar, 37Maerz A.L. Drummer H.E. Wilson K.A. Poumbourios P. J. Virol. 2001; 75: 6635-6644Crossref PubMed Scopus (51) Google Scholar). The introduction of cysteines to the gp41 disulfide-bonded region and to gp120 C1- or C5-generated disulfide-linked gp120-gp41, confirming that these regions are in close contact (38Binley J.M. Sanders R.W. Clas B. Schuelke N. Master A. Guo Y. Kajumo F. Anselma D.J. Maddon P.J. Olson W.C. Moore J.P. J. Virol. 2000; 74: 627-643Crossref PubMed Scopus (451) Google Scholar). The reduction of one such covalently associated gp120-gp41 complex, linked through cysteines introduced at residues 501 of gp120 and 605 of gp41, led to the restoration of cell-cell fusion and entry function indicating that the engineered intersubunit disulfide had formed in a functional Env context (39Abrahamyan L.G. Markosyan R.M. Moore J.P. Cohen F.S. Melikyan G.B. J. Virol. 2003; 77: 5829-5836Crossref PubMed Scopus (62) Google Scholar, 40Binley J.M. Cayanan C.S. Wiley C. Schulke N. Olson W.C. Burton D.R. J. Virol. 2003; 77: 5678-5684Crossref PubMed Scopus (125) Google Scholar).Protein-protein interaction surfaces can exhibit structural plasticity, a mechanism whereby an interface adapts to mutations as binding partners coevolve (41Atwell S. Ultsch M. De Vos A.M. Wells J.A. Science. 1997; 278: 1125-1128Crossref PubMed Scopus (165) Google Scholar). Gp120-gp41 represents an extreme example of a coevolving protein-protein complex with V1, V2, and V3 amino acid sequences diverging by 77, 36, and 46%, respectively, over 7 years in an infected individual, whereas up to 35% sequence divergence has been observed in Env among HIV-1 pandemic strains (24Kuiken C.L. Foley B. Hahn B. Korber B. McCutchan F. Marx P.A. Mellors J.W. Mullins J.I. Sodroski J. Wolinsky S. Human Retroviruses and AIDS 2000: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory, Los Alamos, NV2000Google Scholar, 42Shioda T. Oka S. Xin X. Liu H. Harukuni R. Kurotani A. Fukushima M. Hasan M.K. Shiino T. Takebe Y. Iwamoto A. Nagai Y. J. Virol. 1997; 71: 4871-4881Crossref PubMed Google Scholar). In this study, we examined the functional contribution of 5 conserved gp120 contact residues of the gp41 disulfide-bonded region (37Maerz A.L. Drummer H.E. Wilson K.A. Poumbourios P. J. Virol. 2001; 75: 6635-6644Crossref PubMed Scopus (51) Google Scholar), in the context of Env sequence evolution. Whereas the overall gp120-gp41 association function of the disulfide-bonded region was conserved in Envs from diverse HIV-1 strains, the relative contribution of the individual residues to gp120-anchoring and membrane fusion varied among the strains tested and depended on V1V2 and V3 sequences. These results suggest that the coevolution of gp120 and gp41 is associated with changes to the functional contribution of conserved amino acids in the gp120-gp41 association site.EXPERIMENTAL PROCEDURESHIV-1 Env Expression Vectors—The following HIV-1 molecular clones were used for derivation of pCDNA3.1/myc-HisA+ (Invitrogen)-based env expression vectors: pAD8 (43Theodore T.S. Englund G. Buckler-White A. Buckler C.E. Martin M.A. Peden K.W. AIDS Res. Hum. Retroviruses. 1996; 12: 191-194Crossref PubMed Scopus (120) Google Scholar) from K. W. Peden, CBER, Food and Drug Administration, Bethesda, MD; p89.6 (44Collman R. Balliet J.W. Gregory S.A. Friedman H. Kolson D.L. Nathanson N. Srinivasan A. J. Virol. 1992; 66: 7517-7521Crossref PubMed Google Scholar) from R. G. Collman, University of Pennsylvania School of Medicine, Philadelphia, PA; pSVIII-92UG024.2 and pSVIII-92UG037.8 (45Gao F. Morrison S.G. Robertson D.L. Thornton C.L. Craig S. Karlsson G. Sodroski J. Morgado M. Galvao-Castro B. von Briesen H. Beddows S. Weber J. Sharp P.M. Shaw G.M. Hahn B.H. WHO and NIAID Networks for HIV Isolation and CharacterizationJ. Virol. 1996; 70: 1651-1667Crossref PubMed Google Scholar) from B. Hahn through the National Institutes of Health AIDS Reference and Reagent Program; QH0515.1, QH1549.13, and QH1521.34 (46Hu Q.X. Barry A.P. Wang Z.X. Connolly S.M. Peiper S.C. Greenberg M.L. J. Virol. 2000; 74: 11858-11872Crossref PubMed Scopus (65) Google Scholar) from M. L. Greenberg, Department of Surgery, Duke University Medical Center, Durham, NC; and pTMenv.2, which contains the BH8 env open reading frame (37Maerz A.L. Drummer H.E. Wilson K.A. Poumbourios P. J. Virol. 2001; 75: 6635-6644Crossref PubMed Scopus (51) Google Scholar). The env region was excised from pAD8 with EcoRI and EspI and this fragment was cloned into the EcoRI-EcoRV sites of pCDNA3.1 to give pCDNA3.1-AD8env. The env region of p89.6 was excised using HindIII and EcoRV and then cloned into the corresponding sites of pCDNA3.1 to give pCDNA3.1-89.6env. The KpnI env gene fragments of pSVIII-92UG024.2 and pSVIII-92UG037.8 were cloned into the KpnI sites of pCDNA3.1-89.6env. QH0515.1, QH1549.13, and QH1521.34 KpnI-XbaI env gene fragments were cloned into the corresponding sites in pCDNA3.1-89.6env. Finally, a BH8 env gene fragment was excised from pTMenv.2 using KpnI and SalI and ligated into the KpnI-XhoI sites of pCDNA3.1-89.6env. In vitro mutagenesis was carried out by standard overlap extension PCR techniques. For the sequential isolates, QH0515.1 and QH1549.13, the L593V, W596L, G597A, K601E, and W610F mutations were first introduced into QH1549.13 Env and the wild-type and mutagenized gene fragments encompassing gp41 residues Ser546 to Thr612 used to replace the corresponding region of QH0515.1. BH8-AD8 Env chimeras were made by replacing the V1V2-C2-V3 (DraIII-Bsu36I fragment, AD8V1-3), V1V2 (DraIII-StuI, AD8V1V2), or V3 (AseI-Bsu36I, AD8V3) regions of BH8 with those of AD8 in pcDNA3.1-BH8env. The AD8120 chimera contains AD8 gp120 linked to BH8 gp41 through the common subtilisin cleavage sequence Arg-Glu-Lys-Arg and was constructed using overlap extension PCR. The sequences of mutated Env regions were confirmed by automated DNA sequencing using the ABI prism method.Cells—293T and BHK-21 cells were maintained in Dulbecco's modification of minimal essential medium, 10% fetal calf serum (complete medium). The cells (250,000 cells per 4.5-mm2 well of Linbro 12-well culture dishes, ICN Biomedicals Inc. Aurora, OH) were transfected by the FuGENE 6 procedure (Roche Applied Science). U87 astroglioma cells stably expressing CD4 and CCR5 (U87.CD4.CCR5; obtained from the AIDS Research and Reference Reagent Program from H. Deng and D. Littman (47Bjorndal A. Deng H. Jansson M. Fiore J.R. Colognesi C. Karlsson A. Albert J. Scarlatti G. Littman D.R. Fenyo E.M. J. Virol. 1997; 71: 7478-7487Crossref PubMed Google Scholar)) were maintained in complete medium supplemented with 1 μg/ml puromycin and 300 μg/ml G418.Antibodies—Immunoglobulin G14 (IgG#14) was purified from the plasma of an HIV-1-positive individual using protein A-Sepharose (Amersham Biosciences). The anti-gp41 monoclonal antibody C8 (48Abacioglu Y.H. Fouts T.R. Laman J.D. Claassen E. Pincus S.H. Moore J.P. Roby C.A. Kamin-Lewis R. Lewis G.K. AIDS Res. Hum. Retroviruses. 1994; 10: 371-381Crossref PubMed Scopus (159) Google Scholar) was obtained from G. Lewis through the AIDS Research and Reference Reagent Program, NIAID National Institutes for Health, whereas mAb 2F5 (49Muster T. Steindl F. Purtscher M. Trkola A. Klima A. Himmler G. Ruker F. Katinger H. J. Virol. 1993; 67: 6642-6647Crossref PubMed Google Scholar) was obtained from Polymun Scientific (Vienna, Austria). The polyclonal anti-gp120 antibody DV-012 was obtained from the AIDS Research and Reference Reagent Program, NIAID, from M. Phelan (50Hatch W.C. Tanaka K.E. Calvelli T. Rashbaum W.K. Kress Y. Lyman W.D. J. Immunol. 1992; 148: 3055-3061PubMed Google Scholar).Western Blotting—At 36 h post-transfection, 293T cells were lysed for 10 min on ice in phosphate-buffered saline (PBS) containing 1% Triton X-100, 0.02% sodium azide, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 5 μg ml-1 aprotinin, 5 μg ml-1 leupeptin, 1 mm dithiothreitol. Lysates were clarified by centrifugation at 10,000 × g at 4 °C prior to 10% polyacrylamide gel electrophoresis in the presence of SDS under reducing conditions. Proteins were transferred to nitrocellulose prior to Western blotting with mAb C8 or mAb 2F5 using the enhanced chemiluminescence procedure (Roche Applied Science).Biosynthetic Labeling and Immunoprecipitation—At 16 h post-transfection, 293T cells were incubated for 30 min in cysteine- and methionine-deficient medium (ICN, Costa Mesa, CA), and then labeled for 45 min with 150 μCi of Tran35S-label (ICN). The cells were then washed and chased in complete medium for 4.5 h prior to lysis. In most experiments, incubations were performed at 37 °C. However, for 92UG037, QH1521#34, QH0515#1, and QH1549#13, the pulse and chase were performed at 35 °C for enhanced gp160 processing. Cell lysates and clarified culture supernatants were immunoprecipitated as described previously (51Poumbourios P. Wilson K.A. Center R.J. El Ahmar W. Kemp B.E. J. Virol. 1997; 71: 2041-2049Crossref PubMed Google Scholar) using IgG#14.Surface Biotinylation—At 24 h post-transfection, 293T cells were chilled on ice, washed twice with ice-cold PBS and then incubated with 1.25 mm sulfo-NHS long chain biotin (Pierce) in PBS for 30 min on ice. The biotinylated cells were washed once with ice-cold PBS and quenched with 50 mm glycine/PBS, pH 8.0 (500 μl), for 30 min on ice. The cells were lysed by the addition of 170 μl of ice-cold 4% Triton X-100, 2.4 m KCl, 0.2 m Tris·HCl, pH 7.4, clarified by centrifugation at 10,000 × g for 10 min at 4 °C, and precleared with bovine serum albumin-Sepharose CL-4B for 16 h at 4 °C. Envelope proteins were immunoprecipitated from the precleared cell lysates with IgG#14 and protein G-Sepharose (Amersham Biosciences). Biotinylated Env proteins were visualized following SDS-PAGE on 4-12% polyacrylamide gradient gels under reducing conditions, transfer to nitrocellulose and blotting with neutravidin-horseradish peroxidase (Pierce) and chemiluminescence (Roche Applied Science).Luciferase Reporter Assay for Cell to Cell Fusion—Luciferase reporter assays for cell to cell fusion were performed as described with minor modifications (37Maerz A.L. Drummer H.E. Wilson K.A. Poumbourios P. J. Virol. 2001; 75: 6635-6644Crossref PubMed Scopus (51) Google Scholar). 293T effector cells were transfected with Env-expression constructs and then infected with the recombinant vaccinia virus vTF7.3 at 5 h post-transfection for expression of T7 RNA polymerase (52Moss B. Elroy-Stein O. Mizukami T. Alexander W.A. Fuerst T.R. Nature. 1990; 348: 91-92Crossref PubMed Scopus (448) Google Scholar). BHK-21 or 293T target cells were cotransfected with pT4luc (a dual expression vector for cytomegalovirus promoter-driven CD4 expression and bacteriophage T7 promoter-driven firefly luciferase expression (37Maerz A.L. Drummer H.E. Wilson K.A. Poumbourios P. J. Virol. 2001; 75: 6635-6644Crossref PubMed Scopus (51) Google Scholar)) and a chemokine receptor expression construct: pc.FUSIN, pc.CKR5, or pc.CCR3 (9Deng H. Liu R. Ellmeier W. Choe S. Unutmaz D. Burkhart M. Di Marzio P. Marmon S. Sutton R.E. Hill C.M. Davis C.B. Peiper S.C. Schall T.J. Littman D.R. Landau N.R. Nature. 1996; 381: 661-666Crossref PubMed Scopus (3189) Google Scholar) obtained from Dr. N. Landau through the National Institutes of Health AIDS Research and Reference Reagent Program. At 24 h post-transfection, effectors and targets were resuspended in 1 ml of complete medium containing 1 μg/ml actinomycin D and 40 μg/ml cytosine arabinoside and cocultured for a further 3 (BHK-21) or 16 h (293T) at 37 °C. The cells were then lysed and assayed for luciferase activity using the Promega luciferase assay system.Single Cycle Infectivity Assays—Env-pseudotyped luciferase reporter viruses were produced by cotransfecting 293T cells (350,000 cells per well of a 6-well Linbro culture plate) with 1 μg of Env expression vector and 1 μg of luciferase reporter virus vector pNL4.3.Luc.R-E- (obtained from the AIDS Research and Reference Reagent Program from N. Landau (53Connor R.I. Chen B.K. Choe S. Landau N.R. Virology. 1995; 206: 935-944Crossref PubMed Scopus (1086) Google Scholar)). Supernatants containing pseudotyped virions were harvested at 72 h post-transfection and filtered through 0.45-μm filters. The ability of wild type and mutated Env to mediate infection via CCR5 was assessed in U87.CD4.CCR5 cells. The cells (60,000 cells per well of a 48-well plate, Greiner Labtek) were infected with 200 μl of virus-containing supernatant for 18 h, after which the inoculum was replaced with 500 μl of complete medium. At 52 h postinfection, the cells were assayed with the Promega luciferase assay system using a Berthold Microlumat LB luminometer. The ability of wild-type and mutated Env to mediate infection via CXCR4 was assessed in 293T cells (250,000 cells per well of a 12 well plate) that had been transiently transfected with 1 μg of pc.FUSIN and 1 μg of pCDNA.T4 24 h previously (37Maerz A.L. Drummer H.E. Wilson K.A. Poumbourios P. J. Virol. 2001; 75: 6635-6644Crossref PubMed Scopus (51) Google Scholar). The cells were infected with 800 μl of pseudotyped virus-containing supernatant for 18 h after which the virus inoculum was replaced with 2 ml of complete medium. The cells were assayed for luciferase activity at 50 h postinfection. The protein composition of pseudotyped virions was assessed by first pelleting the virions from 9 ml of culture supernatant through a 1.5-ml 25% (w/v) sucrose/PBS cushion (Beckman SW41 Ti rotor, 25,000 rpm, 2.5 h, 4 °C) followed by reducing SDS-PAGE in 7.5-15% polyacrylamide gradient gels and Western blotting with IgG#14 and DV-012.RESULTSPreviously, we identified five conserved residues, Leu593, Trp596, Gly597, Lys601, and Trp610, in the disulfide-bonded region of gp41 that are important for association with gp120 for a T cell line-adapted (TCLA) HIV-1 isolate BH8. We reasoned that these residues cluster in the gp120 association site and are likely to play a role in receptor-induced conformational changes in gp120-gp41, leading to gp41 fusion activation (Fig. 1A) (37Maerz A.L. Drummer H.E. Wilson K.A. Poumbourios P. J. Virol. 2001; 75: 6635-6644Crossref PubMed Scopus (51) Google Scholar). The functional role of these residues may differ in primary strains as the gp120-gp41 complex of such strains exhibit greater stability and resistance to neutralization with soluble CD4 (sCD4) when compared with TCLA HIV-1 (54Moore J.P. McKeating J.A. Huang Y.X. Ashkenazi A. Ho D.D. J. Virol. 1992; 66: 235-243Crossref PubMed Google Scholar). Subtle functional differences in gp120-gp41 of primary strains relating to chemokine receptor specificity have also been observed. For example, suboptimal sCD4 concentrations lead to enhanced entry by R5 virus

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