scholarly journals Antigenic Properties of the Human Immunodeficiency Virus Envelope during Cell-Cell Fusion

2001 ◽  
Vol 75 (22) ◽  
pp. 11096-11105 ◽  
Author(s):  
Catherine M. Finnegan ◽  
Werner Berg ◽  
George K. Lewis ◽  
Anthony L. DeVico
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Target Cell ◽  
Binding Domain ◽  
Target Cells ◽  
Coreceptor Binding ◽  
Antigenic Properties ◽  
Immunodeficiency Virus ◽  
Cell Interface ◽  
Cell Cell

ABSTRACT Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIVHXB2 envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4°C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.

scholarly journals Antigenic Properties of the Human Immunodeficiency Virus Transmembrane Glycoprotein during Cell-Cell Fusion

2002 ◽  
Vol 76 (23) ◽  
pp. 12123-12134 ◽  
Author(s):  
Catherine M. Finnegan ◽  
Werner Berg ◽  
George K. Lewis ◽  
Anthony L. DeVico
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Target Cells ◽  
Dye Transfer ◽  
Temperature Dependent ◽  
Gp41 Ectodomain ◽  
Immunodeficiency Virus ◽  
Hiv Envelope ◽  
Soluble Cd4 ◽  
Cell Cell

ABSTRACT Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIVHXB2 envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19°C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.

scholarly journals Actin Cytoskeletal Reorganizations and Coreceptor-Mediated Activation of Rac during Human Immunodeficiency Virus-Induced Cell Fusion

2004 ◽  
Vol 78 (13) ◽  
pp. 7138-7147 ◽  
Author(s):  
S. E. Pontow ◽  
N. Vander Heyden ◽  
S. Wei ◽  
L. Ratner
Keyword(s):  
Human Immunodeficiency Virus ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Actin Filament ◽  
Target Cell ◽  
Syncytium Formation ◽  
Dominant Negative ◽  
Target Cells ◽  
Dependent Manner ◽  
Immunodeficiency Virus

ABSTRACT The membrane fusion events which initiate human immunodeficiency virus type 1 (HIV-1) infection and promote cytopathic syncytium formation in infected cells commence with the binding of the HIV envelope glycoprotein (Env) to CD4 and an appropriate coreceptor. Here, we show that HIV Env-coreceptor interactions activate Rac-1 GTPase and stimulate the actin filament network reorganizations that are requisite components of the cell fusion process. Disrupting actin filament dynamics with jasplakinolide or latrunculin A arrested fusion at a late step in the formation of Env-CD4-coreceptor complexes. Time-lapse confocal microscopy of living cells revealed vigorous activity of actin-based, target cell membrane extensions at the target cell-Env-expressing cell interface. The expression of dominant-negative forms of actin-regulating Rho-family GTPases established that HIV Env-mediated syncytium formation relies on Rac-1 but not on Cdc42 or Rho activation in target cells. Similar dependencies were found when cell fusion was induced by Env expressed on viral or cellular membranes. Additionally, Rac activity was specifically upregulated in a coreceptor-dependent manner in fusion reaction cell lysates. These results define a role for HIV Env-coreceptor interactions in activating the cellular factors essential for virus-cell and cell-cell fusion and provide evidence for the participation of pertussis toxin-insensitive signaling pathways in HIV-induced membrane fusion.

scholarly journals Cyanovirin-N Binds to gp120 To Interfere with CD4-Dependent Human Immunodeficiency Virus Type 1 Virion Binding, Fusion, and Infectivity but Does Not Affect the CD4 Binding Site on gp120 or Soluble CD4-Induced Conformational Changes in gp120

1999 ◽  
Vol 73 (5) ◽  
pp. 4360-4371 ◽  
Author(s):  
Mark T. Esser ◽  
Toshiyuki Mori ◽  
Isabelle Mondor ◽  
Quentin J. Sattentau ◽  
Barna Dey ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Conformational Changes ◽  
Target Cell ◽  
Envelope Glycoprotein ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Cd4 Binding Site ◽  
Hiv 1 ◽  
Soluble Cd4

ABSTRACT Cyanovirin-N (CV-N), an 11-kDa protein isolated from the cyanobacterium Nostoc ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. While it has been well established that the viral surface envelope glycoprotein gp120 is a molecular target of CV-N, the detailed mechanism of action is of further interest. We compared matched native and CV-N-treated virus preparations in a panel of assays that measure viral replication, assessing successive stages of the viral life cycle. CV-N-treated virions failed to infect cells as detected by p24 production and quantitative PCR for HIV-1 reverse transcription products, whereas treatment of the target cells did not block infection, confirming that CV-N acts at the level of the virus, not the target cell, to abort the initial infection process. Compared to native HIV-1 preparations, CV-N-treated HIV-1 virions showed impaired CD4-dependent binding to CD4+ T cells and did not mediate “fusion from without” of CD4+ target cells. CV-N also blocked HIV envelope glycoprotein Env-induced, CD4-dependent cell-cell fusion. Mapping studies with monoclonal antibodies (MAbs) to defined epitopes on the HIV-1 envelope glycoprotein indicated that CV-N binds to gp120 in a manner that does not occlude or alter the CD4 binding site or V3 loop or other domains on gp120 recognized by defined MAbs and does not interfere with soluble CD4-induced conformational changes in gp120. Binding of CV-N to soluble gp120 or virions inhibited subsequent binding of the unique neutralizing MAb 2G12, which recognizes a glycosylation-dependent epitope. However, prior binding of 2G12 MAb to gp120 did not block subsequent binding by CV-N. These results help clarify the mechanism of action of CV-N and suggest that the compound may act in part by preventing essential interactions between the envelope glycoprotein and target cell receptors. This proposed mechanism is consistent with the extensive activity profile of CV-N against numerous isolates of HIV-1 and other lentiviruses and supports the potential broad utility of this protein as a microbicide to prevent the sexual transmission of HIV.

scholarly journals Nuclear Import Defect of Human Immunodeficiency Virus Type 1 DNA Flap Mutants Is Not Dependent on the Viral Strain or Target Cell Type

2006 ◽  
Vol 80 (20) ◽  
pp. 10262-10269 ◽  
Author(s):  
Nathalie Arhel ◽  
Sandie Munier ◽  
Philippe Souque ◽  
Karine Mollier ◽  
Pierre Charneau
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cell ◽  
Nuclear Import ◽  
Target Cells ◽  
Virus Infectivity ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have previously established, using human immunodeficiency virus type 1 (HIV-1) strain LAI, that the HIV-1 central DNA Flap acts as a cis determinant of viral genome nuclear import. Although the impact of the DNA Flap on nuclear import has already found numerous independent confirmations in the context of lentivirus vectors, it has been claimed that it may be nonessential for infectious virus strains LAI, YU-2 (J. D. Dvorin et al., J. Virol. 76:12087-12096, 2002), HXB2, and NL4-3 (A. Limon et al., J. Virol. 76:12078-12086, 2002). We conducted a detailed analysis of virus infectivity using the provirus clones provided by the authors and analogous target cells. In contrast to published data, our results show that all cPPT mutant viruses exhibit reduced infectivity corresponding to a nuclear import defect irrespective of the viral genetic background or target cell.

scholarly journals Formation of Syncytia Is Repressed by Tetraspanins in Human Immunodeficiency Virus Type 1-Producing Cells

2009 ◽  
Vol 83 (15) ◽  
pp. 7467-7474 ◽  
Author(s):  
Jia Weng ◽  
Dimitry N. Krementsov ◽  
Sandhya Khurana ◽  
Nathan H. Roy ◽  
Markus Thali
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Syncytium Formation ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In vitro propagation studies have established that human immunodeficiency virus type 1 (HIV-1) is most efficiently transmitted at the virological synapse that forms between producer and target cells. Despite the presence of the viral envelope glycoprotein (Env) and CD4 and chemokine receptors at the respective surfaces, producer and target cells usually do not fuse with each other but disengage after the viral particles have been delivered, consistent with the idea that syncytia, at least in vitro, are not required for HIV-1 spread. Here, we tested whether tetraspanins, which are well known regulators of cellular membrane fusion processes that are enriched at HIV-1 exit sites, regulate syncytium formation. We found that overexpression of tetraspanins in producer cells leads to reduced syncytium formation, while downregulation has the opposite effect. Further, we document that repression of Env-induced cell-cell fusion by tetraspanins depends on the presence of viral Gag, and we demonstrate that fusion repression requires the recruitment of Env by Gag to tetraspanin-enriched microdomains (TEMs). However, sensitivity to fusion repression by tetraspanins varied for different viral strains, despite comparable recruitment of their Envs to TEMs. Overall, these data establish tetraspanins as negative regulators of HIV-1-induced cell-cell fusion, and they start delineating the requirements for this regulation.

scholarly journals Rational Site-Directed Mutations of the LLP-1 and LLP-2 Lentivirus Lytic Peptide Domains in the Intracytoplasmic Tail of Human Immunodeficiency Virus Type 1 gp41 Indicate Common Functions in Cell-Cell Fusion but Distinct Roles in Virion Envelope Incorporation

2003 ◽  
Vol 77 (6) ◽  
pp. 3634-3646 ◽  
Author(s):  
Vandana Kalia ◽  
Surojit Sarkar ◽  
Phalguni Gupta ◽  
Ronald C. Montelaro
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Syncytium Formation ◽  
Immunodeficiency Virus ◽  
Arginine Residues ◽  
Hiv 1 ◽  
Cell Cell

ABSTRACT Two highly conserved cationic amphipathic α-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses . Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.

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