Sequential CD4-Coreceptor Interactions in Human Immunodeficiency Virus Type 1 Env Function: Soluble CD4 Activates Env for Coreceptor-Dependent Fusion and Reveals Blocking Activities of Antibodies against Cryptic Conserved Epitopes on gp120

ABSTRACT We devised an experimental system to examine sequential events by which the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) interacts with CD4 and coreceptor to induce membrane fusion. Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems.

[1]  B. Moss,et al.  Compact, synthetic, vaccinia virus early/late promoter for protein expression. , 1997, BioTechniques.

[2]  J. Robinson,et al.  Resistance of human immunodeficiency virus type 1 to neutralization by natural antisera occurs through single amino acid substitutions that cause changes in antibody binding at multiple sites , 1996, Journal of virology.

[3]  B. Moss,et al.  Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor , 1992, Journal of virology.

[4]  P. Earl,et al.  Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities , 1994, Journal of virology.

[5]  D. Littman,et al.  Fusion-competent vaccines: broad neutralization of primary isolates of HIV. , 1999, Science.

[6]  P. S. Kim,et al.  HIV Entry and Its Inhibition , 1998, Cell.

[7]  R. M. Hendry,et al.  Neutralization of primary HIV‐1 isolates by anti‐envelope monoclonal antibodies , 1995, AIDS.

[8]  R. Doms,et al.  HIV and SIV gp120 binding does not predict coreceptor function. , 1999, Virology.

[9]  T L Hoffman,et al.  Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Susan Zolla-Pazner,et al.  Human Immunodeficiency Virus (HIV) Envelope Binds to CXCR4 Independently of CD4, and Binding Can Be Enhanced by Interaction with Soluble CD4 or by HIV Envelope Deglycosylation , 1998, Journal of Virology.

[11]  C. Broder,et al.  Fusogenic selectivity of the envelope glycoprotein is a major determinant of human immunodeficiency virus type 1 tropism for CD4+ T-cell lines vs. primary macrophages. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  C. Barbas,et al.  Determinants of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Activation by Soluble CD4 and Monoclonal Antibodies , 1998, Journal of Virology.

[13]  D. Dimitrov,et al.  Evidence for Cell-Surface Association Between Fusin and the CD4-gp120 Complex in Human Cell Lines , 1996, Science.

[14]  J. Sodroski,et al.  Effects of Soluble CD4 on Simian Immunodeficiency Virus Infection of CD4-Positive and CD4-Negative Cells , 1999, Journal of Virology.

[15]  J. Berzofsky,et al.  Induction of a Mucosal Cytotoxic T-Lymphocyte Response by Intrarectal Immunization with a Replication-Deficient Recombinant Vaccinia Virus Expressing Human Immunodeficiency Virus 89.6 Envelope Protein , 1998, Journal of Virology.

[16]  J. Allan,et al.  Enhancement of SIV infection with soluble receptor molecules. , 1990, Science.

[17]  S. Zolla-Pazner,et al.  Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor , 1997, Journal of virology.

[18]  Q. Sattentau,et al.  HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4. , 1997, Journal of immunology.

[19]  J. Hoxie,et al.  CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons , 1997, Current Biology.

[20]  J. Sodroski,et al.  CD4-Induced Conformational Changes in the Human Immunodeficiency Virus Type 1 gp120 Glycoprotein: Consequences for Virus Entry and Neutralization , 1998, Journal of Virology.

[21]  C. Broder,et al.  Phorbol ester-induced down modulation of tailless CD4 receptors requires prior binding of gp120 and suggests a role for accessory molecules , 1995, Journal of virology.

[22]  Paul E. Kennedy,et al.  HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein-Coupled Receptor , 1996, Science.

[23]  J. Sodroski,et al.  Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization , 1996, Journal of virology.

[24]  Stephen C. Peiper,et al.  Identification of CXCR4 Domains That Support Coreceptor and Chemokine Receptor Functions , 1999, Journal of Virology.

[25]  Peter D. Kwong,et al.  The antigenic structure of the HIV gp120 envelope glycoprotein , 1998, Nature.

[26]  J. Sodroski,et al.  The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. , 1998, Science.

[27]  H. Guy,et al.  Epitope Mapping of CCR5 Reveals Multiple Conformational States and Distinct but Overlapping Structures Involved in Chemokine and Coreceptor Function* , 1999, The Journal of Biological Chemistry.

[28]  J. Sodroski,et al.  Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding , 1993, Journal of virology.

[29]  D. Missé,et al.  A CD4-independent interaction of human immunodeficiency virus-1 gp120 with CXCR4 induces their cointernalization, cell signaling, and T-cell chemotaxis. , 1999, Blood.

[30]  J. Sodroski,et al.  Interactions among HIV gp120, CD4, and CXCR4: dependence on CD4 expression level, gp120 viral origin, conservation of the gp120 COOH- and NH2-termini and V1/V2 and V3 loops, and sensitivity to neutralizing antibodies. , 1998, Virology.

[31]  J. Moore,et al.  Exploration of antigenic variation in gp120 from clades A through F of human immunodeficiency virus type 1 by using monoclonal antibodies , 1994, Journal of virology.

[32]  Q. Sattentau,et al.  Neutralization of Human Immunodeficiency Virus Type 1 by Antibody to gp120 Is Determined Primarily by Occupancy of Sites on the Virion Irrespective of Epitope Specificity , 1998, Journal of Virology.

[33]  R. Weiss,et al.  Human immunodeficiency virus type 2 infection and fusion of CD4-negative human cell lines: induction and enhancement by soluble CD4 , 1992, Journal of virology.

[34]  R. Weiss,et al.  CD4-independent infection by HIV-2 (ROD/B): use of the 7-transmembrane receptors CXCR-4, CCR-3, and V28 for entry. , 1997, Virology.

[35]  Ying Sun,et al.  The β-Chemokine Receptors CCR3 and CCR5 Facilitate Infection by Primary HIV-1 Isolates , 1996, Cell.

[36]  R. Doms,et al.  CD4-Independent Infection by HIV-2 Is Mediated by Fusin/CXCR4 , 1996, Cell.

[37]  Virginia Litwin,et al.  HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5 , 1996, Nature.

[38]  M. L. Penn,et al.  A trans-receptor mechanism for infection of CD4-negative cells by human immunodeficiency virus type 1 , 1999, Current Biology.

[39]  Ying Sun,et al.  A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. , 1998, Science.

[40]  C. Broder,et al.  CC CKR5: A RANTES, MIP-1α, MIP-1ॆ Receptor as a Fusion Cofactor for Macrophage-Tropic HIV-1 , 1996, Science.

[41]  E. Tramont,et al.  The human immunodeficiency virus. , 1991, Dermatologic clinics.

[42]  J. Sodroski,et al.  Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding , 1995, Journal of virology.

[43]  J. Allan.  Receptor-mediated activation of the viral envelope and viral entry. , 1993, AIDS.

[44]  S. Harrison,et al.  Structural basis for membrane fusion by enveloped viruses. , 1999, Molecular membrane biology.

[45]  J. Sodroski,et al.  Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody , 1998, Nature.

[46]  William C. Olson,et al.  CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5 , 1996, Nature.

[47]  D. Missé,et al.  Dissociation of the CD4 and CXCR4 Binding Properties of Human Immunodeficiency Virus Type 1 gp120 by Deletion of the First Putative Alpha-Helical Conserved Structure , 1998, Journal of Virology.

[48]  J. Farber,et al.  Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. , 1999, Annual review of immunology.

[49]  J. Sodroski,et al.  CD4-independent binding of SIV gp120 to rhesus CCR5. , 1997, Science.

[50]  Stephen C. Peiper,et al.  Identification of a major co-receptor for primary isolates of HIV-1 , 1996, Nature.

[51]  J. Sodroski,et al.  Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by a gp41 amino acid change , 1994, Journal of virology.

[52]  J. Sodroski,et al.  Antigenic variation in gp120s from molecular clones of HIV-1 LAI. , 1993, AIDS research and human retroviruses.

[53]  Marc Parmentier,et al.  A Dual-Tropic Primary HIV-1 Isolate That Uses Fusin and the β-Chemokine Receptors CKR-5, CKR-3, and CKR-2b as Fusion Cofactors , 1996, Cell.

[54]  C. Broder,et al.  Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation , 1994, Journal of virology.

[55]  James E. K. Hildreth,et al.  T cell-tropic HIV gp120 mediates CD4 and CD8 cell chemotaxis through CXCR4 independent of CD4: implications for HIV pathogenesis. , 1999, Journal of immunology.

[56]  R. Doms,et al.  Functional Dissection of CCR5 Coreceptor Function through the Use of CD4-Independent Simian Immunodeficiency Virus Strains , 1999, Journal of Virology.

[57]  Q. Sattentau,et al.  Probing the structure of the V2 domain of human immunodeficiency virus type 1 surface glycoprotein gp120 with a panel of eight monoclonal antibodies: human immune response to the V1 and V2 domains , 1993, Journal of virology.

[58]  P. Sharp,et al.  Genetic variation of HIV type 1 in four World Health Organization-sponsored vaccine evaluation sites: generation of functional envelope (glycoprotein 160) clones representative of sequence subtypes A, B, C, and E. WHO Network for HIV Isolation and Characterization. , 1994, AIDS research and human retroviruses.

[59]  Joseph Sodroski,et al.  CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5 , 1996, Nature.

[60]  Q. Sattentau,et al.  Constitutive cell surface association between CD4 and CCR5. , 1999, Proceedings of the National Academy of Sciences of the United States of America.