scholarly journals Stabilization of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers by Disulfide Bonds Introduced into the gp41 Glycoprotein Ectodomain

1998 ◽  
Vol 72 (9) ◽  
pp. 7620-7625 ◽  
Author(s):  
Michael Farzan ◽  
Hyeryun Choe ◽  
Elizabeth Desjardins ◽  
Ying Sun ◽  
Jens Kuhn ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Disulfide Bonds ◽  
Coiled Coil ◽  
Alpha Helices ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Biochemical and structural studies of fragments of the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein have demonstrated that the molecular contacts between alpha helices allow the formation of a trimeric coiled coil. By introducing cysteine residues into specific locations along these alpha helices, the normally labile HIV-1 gp160 envelope glycoprotein was converted into a stable disulfide-linked oligomer. Although proteolytic cleavage into gp120 and gp41 glycoproteins was largely blocked, the disulfide-linked oligomer was efficiently transported to the cell surface and was recognized by a series of conformationally dependent antibodies. The pattern of hetero-oligomer formation between this construct and an analogous construct lacking portions of the gp120 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers. These results support the relevance of the proposed gp41 structure and intersubunit contacts to the native, complete HIV-1 envelope glycoprotein. Disulfide-mediated stabilization of the labile HIV-1 envelope glycoprotein oligomer, which has been suggested to possess advantages as an immunogen, may assist attempts to develop vaccines.

scholarly journals Highly Stable Trimers Formed by Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Fused with the Trimeric Motif of T4 Bacteriophage Fibritin

2002 ◽  
Vol 76 (9) ◽  
pp. 4634-4642 ◽  
Author(s):  
Xinzhen Yang ◽  
Juliette Lee ◽  
Erin M. Mahony ◽  
Peter D. Kwong ◽  
Richard Wyatt ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Neutralizing Antibodies ◽  
Envelope Glycoproteins ◽  
Immunodeficiency Virus ◽  
T4 Bacteriophage ◽  
Hiv 1

ABSTRACT The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane proteins. Soluble gp140 glycoproteins composed of the uncleaved ectodomains of gp120 and gp41 form unstable, heterogeneous oligomers, but soluble gp140 trimers can be stabilized by fusion with a C-terminal, trimeric GCN4 motif (X. Yang et al., J. Virol. 74:5716-5725, 2000). To understand the influence of the C-terminal trimerization domain on the properties of soluble HIV-1 envelope glycoprotein trimers, uncleaved, soluble gp140 glycoproteins were stabilized by fusion with another trimeric motif derived from T4 bacteriophage fibritin. The fibritin construct was more stable to heat and reducing conditions than the GCN4 construct. Both GCN4- and fibritin-stabilized soluble gp140 glycoproteins exhibited patterns of neutralizing and nonneutralizing antibody binding expected for the functional envelope glycoprotein spike. Of note, two potently neutralizing antibodies, immunoglobulin G1b12 and 2G12, exhibited the greatest recognition of the stabilized, soluble trimers, relative to recognition of the gp120 monomer. The observed similarities between the GCN4 and fibritin constructs indicate that the HIV-1 envelope glycoprotein ectodomains dictate many of the antigenic and structural features of these fusion proteins. The melting temperatures and ligand recognition properties of the GCN4- and fibritin-stabilized soluble gp140 glycoproteins suggest that these molecules assume conformations distinct from that of the fusion-active, six-helix bundle.

scholarly journals Stoichiometry of Antibody Neutralization of Human Immunodeficiency Virus Type 1

2005 ◽  
Vol 79 (6) ◽  
pp. 3500-3508 ◽  
Author(s):  
Xinzhen Yang ◽  
Svetla Kurteva ◽  
Sandra Lee ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Envelope Glycoproteins ◽  
Antibody Neutralization ◽  
Antibody Molecule ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus envelope glycoproteins function as trimers on the viral surface, where they are targeted by neutralizing antibodies. Different monoclonal antibodies neutralize human immunodeficiency virus type 1 (HIV-1) infectivity by binding to structurally and functionally distinct moieties on the envelope glycoprotein trimer. By measuring antibody neutralization of viruses with mixtures of neutralization-sensitive and neutralization-resistant envelope glycoproteins, we demonstrate that the HIV-1 envelope glycoprotein trimer is inactivated by the binding of a single antibody molecule. Virus neutralization requires essentially all of the functional trimers to be occupied by at least one antibody. This model applies to antibodies differing in neutralizing potency and to virus isolates with various neutralization sensitivities. Understanding these requirements for HIV-1 neutralization by antibodies will assist in establishing goals for an effective AIDS vaccine.

scholarly journals Cellular Immune Responses in Asymptomatic Human Immunodeficiency Virus Type 1 (HIV-1) Infection and Effects of Vaccination with Recombinant Envelope Glycoprotein of HIV-1

2006 ◽  
Vol 13 (1) ◽  
pp. 26-32 ◽  
Author(s):  
Geoffrey J. Gorse ◽  
Ramona E. Simionescu ◽  
Gira B. Patel
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Lymphocyte Proliferation ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1

ABSTRACT Effects of human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein vaccines on cell-mediated immune (CMI) responses were assessed in HIV-1-infected patients. Asymptomatic, antiretroviral-treatment-naïve, HIV-1-infected patients with CD4+ T-cell counts greater than 400/μl received multiple intramuscular injections of HIV-1 IIIB recombinant envelope glycoprotein (rgp160) vaccine or HIV-1 MN recombinant envelope glycoprotein (rgp120) vaccine (eight patients, referred to as the HIV-1 vaccinees) or placebo or hepatitis B vaccine (three patients, referred to as the controls). Lymphocyte proliferation in response to HIV-1 envelope glycoproteins, both homologous and heterologous to the HIV-1 immunogens, was absent prior to study treatment in all patients but increased significantly during the vaccination series and after the final vaccination in HIV-1 vaccinees (P < 0.05) and remained absent in control patients. In flow cytometric analyses of intracellular cytokines, T-cell receptor stimulation with an anti-CD3 antibody induced gamma interferon (IFN-γ) expression by activated CD4+ and CD8+ lymphocytes at greater frequencies than did stimulation with recombinant envelope glycoprotein and p24 of HIV-1 (P< 0.05). Mean frequencies of HIV-1 envelope glycoprotein-stimulated, activated intracellularIFN-γ-producing CD4+ and CD8+ lymphocytes and of interleukin-2-producing CD4+ lymphocytes did not increase after vaccination, but cytokine-producing cells were detectable in some patients. Comparing pre- to post-HIV-1 vaccination time points, changes in frequencies of activated, IFN-γ-producing CD4+ cells correlated inversely with changes in lymphocyte proliferation in response to recombinant envelope glycoprotein in HIV-1 vaccinees (P < 0.05). Increased CMI responses to HIV-1 envelope glycoprotein measured by lymphocyte proliferation were associated with HIV-1 recombinant envelope glycoprotein vaccines.

scholarly journals Characterization of Stable, Soluble Trimers Containing Complete Ectodomains of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins

2000 ◽  
Vol 74 (12) ◽  
pp. 5716-5725 ◽  
Author(s):  
Xinzhen Yang ◽  
Michael Farzan ◽  
Richard Wyatt ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cleavage Site ◽  
Neutralizing Antibodies ◽  
Coiled Coil ◽  
Envelope Glycoproteins ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins function as a membrane-anchored trimer of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Previously, we reported three approaches to stabilize soluble trimers containing parts of the gp41 ectodomains: addition of GCN4 trimeric helices, disruption of the cleavage site between gp120 and gp41, and introduction of cysteines in the gp41 coiled coil to form intersubunit disulfide bonds. Here, we applied similar approaches to stabilize soluble gp140 trimers including the complete gp120 and gp41 ectodomains. A combination of fusion with the GCN4 trimeric sequences and disruption of the gp120-gp41 cleavage site resulted in relatively homogeneous gp140 trimers with exceptional stability. The gp120 epitopes recognized by neutralizing antibodies are intact and exposed on these gp140 trimers. By contrast, the nonneutralizing antibody epitopes on the gp120 subunits of the soluble trimers are relatively occluded compared with those on monomeric gp120 preparations. This antigenic similarity to the functional HIV-1 envelope glycoproteins and the presence of the complete gp41 ectodomain should make the soluble gp140 trimers useful tools for structural and immunologic studies.

scholarly journals Characterization of the Outer Domain of the gp120 Glycoprotein from Human Immunodeficiency Virus Type 1

2004 ◽  
Vol 78 (23) ◽  
pp. 12975-12986 ◽  
Author(s):  
Xinzhen Yang ◽  
Vesko Tomov ◽  
Svetla Kurteva ◽  
Liping Wang ◽  
Xinping Ren ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Antibody Responses ◽  
Outer Domain ◽  
Immunodeficiency Virus ◽  
Gp120 Glycoprotein ◽  
Hiv 1

ABSTRACT The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1YU2 gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.

scholarly journals Two N-Linked Glycosylation Sites in the V2 and C2 Regions of Human Immunodeficiency Virus Type 1 CRF01_AE Envelope Glycoprotein gp120 Regulate Viral Neutralization Susceptibility to the Human Monoclonal Antibody Specific for the CD4 Binding Domain

2010 ◽  
Vol 84 (9) ◽  
pp. 4311-4320 ◽  
Author(s):  
Piraporn Utachee ◽  
Shota Nakamura ◽  
Panasda Isarangkura-na-ayuthaya ◽  
Kenzo Tokunaga ◽  
Pathom Sawanpanyalert ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Human Monoclonal Antibody ◽  
Binding Domain ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A recombinant human monoclonal antibody, IgG1 b12 (b12), recognizes a conformational epitope on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) gp120 that overlaps the CD4 binding domain. Although b12 is able to broadly neutralize HIV-1 subtype B, C, and D viruses, many HIV-1 CRF01_AE viruses are resistant to b12-mediated neutralization. In this report, we examined the molecular mechanisms underlying the low neutralization susceptibility of CRF01_AE viruses to b12, using recently established CRF01_AE Env recombinant viruses. Our results showed that two potential N-linked glycosylation (PNLG) sites in the V2 and C2 regions of Env gp120 played an important role in regulating the susceptibility of CRF01_AE Env to b12. The locations of these PNLG sites correspond to amino acid positions 186 and 197 in HXB2 Env gp120; thus, they are designated N186 and N197 in this study. Removal of N186 significantly conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones, 65CC4 and 107CC2, while the introduction of N186 reduced the b12 susceptibility of a susceptible CRF01_AE Env clone, 65CC1. In addition, removal of both N186 and N197 conferred the b12 susceptibility of 3 resistant CRF01_AE Env clones, 45PB1, 62PL1, and 101PL1, whereas the removal of either N186 or N197 was not sufficient to confer the b12 susceptibility of these CRF01_AE Env clones. Finally, removal of N197 conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones lacking N186, 55PL1 and 102CC2. Taken together, we propose that two PNLG sites, N186 and N197, in Env gp120 are important determinants of the b12 resistance of CRF01_AE viruses.

scholarly journals Oligomeric and Conformational Properties of a Proteolytically Mature, Disulfide-Stabilized Human Immunodeficiency Virus Type 1 gp140 Envelope Glycoprotein

2002 ◽  
Vol 76 (15) ◽  
pp. 7760-7776 ◽  
Author(s):  
Norbert Schülke ◽  
Mika S. Vesanen ◽  
Rogier W. Sanders ◽  
Ping Zhu ◽  
Min Lu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Viral Envelope ◽  
Antigenic Properties ◽  
Immunodeficiency Virus ◽  
Conformational Properties ◽  
Hiv 1

ABSTRACT We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1JR-FL SOS gp140, proteolytically uncleaved gp140 (gp140UNC), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140UNC, SOS gp140 reacted more strongly in surface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies (MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140UNC displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41ECTO) occludes the “nonneutralizing” face of gp120, consistent with the antigenic properties of this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE), a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140UNC comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140UNC were largely unaffected by purification. An uncleaved gp140 protein containing the SOS cysteine mutations (SOS gp140UNC) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.

scholarly journals Genetic Evidence that Interhelical Packing Interactions in the gp41 Core Are Critical for Transition of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein to the Fusion-Active State

2002 ◽  
Vol 76 (14) ◽  
pp. 7356-7362 ◽  
Author(s):  
Kathryn E. Follis ◽  
Scott J. Larson ◽  
Min Lu ◽  
Jack H. Nunberg
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Conformational Changes ◽  
Envelope Glycoprotein ◽  
Antiviral Drug ◽  
Immunodeficiency Virus ◽  
Packing Interactions ◽  
Hiv 1

ABSTRACT The envelope glycoprotein complex (gp120-gp41) of human immunodeficiency virus type 1 (HIV-1) promotes the fusion of viral and cellular membranes through formation of the fusion-active six-helix bundle in the gp41 ectodomain. This gp41 core structure consists of three C-terminal helices packed in an antiparallel manner into hydrophobic grooves on the surface of the N-terminal trimeric coiled coil. Alanine mutations that destabilize the N- and C-terminal interhelical packing interactions also reduce viral infectivity. Here we show that viruses bearing these mutations exhibit a marked potentiation of inhibition by peptides that make up the gp41 core. By contrast, these viruses are unchanged in their sensitivities to soluble CD4, the CXCR4 coreceptor ligand SDF-1α, and human anti-HIV immunoglobulin, reagents that impact the initial, receptor-induced conformational changes in the envelope glycoprotein. Our results support the notion that these alanine mutations specifically affect the conformational transition to the fusion-active gp41 structure. The mutations also increase viral sensitivity to the gp41-directed monoclonal antibody 2F5, suggesting that this broadly neutralizing antibody may also interfere with this transition. The conformational activation of the HIV-1 envelope glycoprotein likely represents a viable target for vaccine and antiviral drug development.

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