Virus-cell membrane fusion does not predict efficient infection of alveolar macrophages by human immunodeficiency virus type 1 (HIV-1)

ABSTRACT In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-β-cyclodextrin (MβCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1α- and macrophage inflammatory protein 1β-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MβCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.

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Regulation of Human Immunodeficiency Virus Type 1 Env-Mediated Membrane Fusion by Viral Protease Activity

Journal of Virology ◽

10.1128/jvi.78.2.1026-1031.2004 ◽

2004 ◽

Vol 78 (2) ◽

pp. 1026-1031 ◽

Cited By ~ 121

Author(s):

Tsutomu Murakami ◽

Sherimay Ablan ◽

Eric O. Freed ◽

Yuetsu Tanaka

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Cytoplasmic Tail ◽

Target Cells ◽

Gag Protein ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR+) or inactive (PR−) viral PR. We observed that PR− virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.

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Inhibitory Effects of Small-Molecule CCR5 Antagonists on Human Immunodeficiency Virus Type 1 Envelope-Mediated Membrane Fusion and Viral Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3538-3543.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3538-3543 ◽

Cited By ~ 16

Author(s):

Katsunori Takashima ◽

Hiroshi Miyake ◽

Rika A. Furuta ◽

Jun-Ichi Fujisawa ◽

Yuji Iizawa ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Viral Replication ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Small Molecule ◽

Inhibitory Effects ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We established a human immunodeficiency virus type 1 (HIV-1) envelope (Env)-mediated membrane fusion assay and examined the small-molecule CCR5 antagonist TAK-779 and its derivatives for their inhibitory effects on HIV-1 Env-mediated membrane fusion and viral replication. The membrane fusion assay is based on HIV-1 long terminal repeat-directed β-d-galactosidase reporter gene expression in CD4- and CCR5-expressed HeLa (MAGI-CCR5) cells after cocultivation with effector 293T cells expressing HIV-1 Env. Inhibition of HIV-1 replication was also determined in MAGI-CCR5 cells infected with the corresponding cell-free HIV-1. TAK-779 effectively suppressed R5 HIV-1 (strain JR-FL) Env-mediated membrane fusion as well as viral replication. Its 50% inhibitory concentrations (IC50s) for membrane fusion and viral replication were 0.87 ± 0.11 and 1.4 ± 0.1 nM, respectively. These values corresponded well to the IC50 for 125I-RANTES (regulated on activation, T cell expressed, and secreted) binding to CCR5 (1.4 nM). The inhibitory effects of 18 TAK-779 derivatives on membrane fusion differed from one compound to another. However, there was a close correlation among their inhibitory effects on membrane fusion, viral replication, and RANTES binding. The correlation coefficient between their IC50s for membrane fusion and viral replication was 0.881. Furthermore, since this assay depends on Env expressed in the effector cells, it is also applicable to the evaluation of CXCR4 antagonists. These results indicate that the HIV-1 Env-mediated membrane fusion assay is a useful tool for the evaluation of entry inhibitors.

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Subdomain Folding and Biological Activity of the Core Structure from Human Immunodeficiency Virus Type 1 gp41: Implications for Viral Membrane Fusion

Journal of Virology ◽

10.1128/jvi.73.5.4433-4438.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4433-4438 ◽

Cited By ~ 44

Author(s):

Min Lu ◽

Hong Ji ◽

Steven Shen

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Covalent Attachment ◽

Direct Role ◽

The Core ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of two subunits, gp120 and gp41. The extraviral portion (ectodomain) of gp41 contains an α-helical domain that likely represents the core of the fusion-active conformation of the molecule. Here we report the identification and characterization of a minimal, autonomous folding subdomain that retains key determinants in specifying the overall fold of the gp41 ectodomain core. This subdomain, designated N34(L6)C28, is formed by covalent attachment of peptides N-34 and C-28 by a short flexible linker in place of the normal disulfide-bonded loop sequence. N34(L6)C28 forms a highly thermostable, α-helical trimer. Point mutations within the envelope protein complex that abolish membrane fusion and HIV-1 infectivity also impede the formation of the N34(L6)C28 core. Moreover, N34(L6)C28 is capable of inhibiting HIV-1 envelope-mediated membrane fusion. Taken together, these results indicate that the N34(L6)C28 core plays a direct role in the membrane fusion step of HIV-1 infection and thus provides a molecular target for the development of antiviral pharmaceutical agents.

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A Conformation-Specific Monoclonal Antibody Reacting with Fusion-Active gp41 from the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Journal of Virology ◽

10.1128/jvi.72.12.10213-10217.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 10213-10217 ◽

Cited By ~ 122

Author(s):

Shibo Jiang ◽

Kang Lin ◽

Min Lu

Keyword(s):

Monoclonal Antibody ◽

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Envelope Glycoprotein ◽

Structural Domain ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays a major role in the membrane fusion step of viral infection. The ectodomain of gp41 contains a six-helix structural domain that likely represents the core of the fusion-active conformation of the molecule. A monoclonal antibody (MAb), designated NC-1, was generated and cloned from a mouse immunized with the model polypeptide N36(L6)C34, which folds into a stable six-helix bundle. NC-1 binds specifically to both the α-helical core domain and the oligomeric forms of gp41. This conformation-dependent reactivity is dramatically reduced by point mutations within the N-terminal coiled-coil region of gp41 which impede formation of the gp41 core. NC-1 binds to the surfaces of HIV-1-infected cells only in the presence of soluble CD4. These results indicate that NC-1 is capable of reacting with fusion-active gp41 in a conformation-specific manner and can be used as a valuable biological reagent for studying the receptor-induced conformational changes in gp41 required for membrane fusion and HIV-1 infection.

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Stoichiometry of Envelope Glycoprotein Trimers in the Entry of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.79.19.12132-12147.2005 ◽

2005 ◽

Vol 79 (19) ◽

pp. 12132-12147 ◽

Cited By ~ 125

Author(s):

Xinzhen Yang ◽

Svetla Kurteva ◽

Xinping Ren ◽

Sandra Lee ◽

Joseph Sodroski

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Influenza A Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Influenza A ◽

Virus Entry ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Envs) function as a trimer, mediating virus entry by promoting the fusion of the viral and target cell membranes. HIV-1 Env trimers induce membrane fusion through a pH-independent pathway driven by the interaction between an Env trimer and its cellular receptors, CD4 and CCR5/CXCR4. We studied viruses with mixed heterotrimers of wild-type and dominant-negative Envs to determine the number (T) of Env trimers required for HIV-1 entry. To our surprise, we found that a single Env trimer is capable of supporting HIV-1 entry; i.e., T = 1. A similar approach was applied to investigate the entry stoichiometry of envelope glycoproteins from amphotropic murine leukemia virus (A-MLV), avian sarcoma/leukosis virus type A (ASLV-A), and influenza A virus. When pseudotyped on HIV-1 virions, the A-MLV and ASLV-A Envs also exhibit a T = 1 entry stoichiometry. In contrast, eight to nine influenza A virus hemagglutinin trimers function cooperatively to achieve membrane fusion and virus entry, using a pH-dependent pathway. The different entry requirements for cooperativity among Env trimers for retroviruses and influenza A virus may influence viral strategies for replication and evasion of the immune system.

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Mutagenesis of CXCR4 Identifies Important Domains for Human Immunodeficiency Virus Type 1 X4 Isolate Envelope-Mediated Membrane Fusion and Virus Entry and Reveals Cryptic Coreceptor Activity for R5 Isolates

Journal of Virology ◽

10.1128/jvi.73.8.6598-6609.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6598-6609 ◽

Cited By ~ 67

Author(s):

Donald J. Chabot ◽

Peng-Fei Zhang ◽

Gerald V. Quinnan ◽

Christopher C. Broder

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Conformational Changes ◽

Alanine Scanning Mutagenesis ◽

Gene Assay ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4’s coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.

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Functional Chimeras of Human Immunodeficiency Virus Type 1 gp120 and Influenza A Virus (H3) Hemagglutinin

Journal of Virology ◽

10.1128/jvi.79.10.6459-6471.2005 ◽

2005 ◽

Vol 79 (10) ◽

pp. 6459-6471 ◽

Cited By ~ 2

Author(s):

K. M. Copeland ◽

A. J. Elliot ◽

R. S. Daniels

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Therapeutic Vaccine ◽

Chimeric Protein ◽

Influenza Viruses ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT In an attempt to produce a protein that will allow determination of the native human immunodeficiency virus type 1 (HIV-1) gp120 (Env) structure in its trimeric state, we fused the globular head of gp120 to the stalk region of influenza virus A (X31) hemagglutinin (HA). The chimeric protein (EnvHA) has been expressed by using a recombinant vaccinia virus system, and its functional characteristics were determined. EnvHA is expressed as a 120- to 150-kDa protein that can oligomerize to form dimers and trimers. It retains the low-pH (5.2 to 5.4) requirement of X31-HA to trigger membrane fusion but, unlike X31-HA, it is not absolutely dependent on exogenously added trypsin for protein processing to release the HA2 fusion peptide. In terms of receptor binding the chimeric protein retains specificity for human CD4 but, in relation to the membrane fusion event, it appears to lose the Env coreceptor specificity of the parental HIV-1 strains: NL43 for CXCR4 and JRFL for CCR5. These properties suggest that stable, functional EnvHAs are being produced and that they may be exploited in terms of structural studies. Further, the potential of introducing the envHA genes into influenza viruses, by use of reverse genetics, and their use as a therapeutic vaccine for HIV are discussed.

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Inhibition of Human Immunodeficiency Virus Type 1 Infectivity by the gp41 Core: Role of a Conserved Hydrophobic Cavity in Membrane Fusion

Journal of Virology ◽

10.1128/jvi.73.10.8578-8586.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8578-8586 ◽

Cited By ~ 55

Author(s):

Hong Ji ◽

Wei Shu ◽

F. Temple Burling ◽

Shibo Jiang ◽

Min Lu

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Coiled Coil ◽

Helix Bundle ◽

Hydrophobic Cavity ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The gp41 envelope protein of human immunodeficiency virus type 1 (HIV-1) contains an α-helical core structure responsible for mediating membrane fusion during viral entry. Recent studies suggest that a conserved hydrophobic cavity in the coiled coil of this core plays a distinctive structural role in maintaining the fusogenic conformation of the gp41 molecule. Here we investigated the importance of this cavity in determining the structure and biological activity of the gp41 core by using the N34(L6)C28 model. The high-resolution crystal structures of N34(L6)C28 of two HIV-1 gp41 fusion-defective mutants reveal that each mutant sequence is accommodated in the six-helix bundle structure by forming the cavity with different sets of atoms. Remarkably, the mutant N34(L6)C28 cores are highly effective inhibitors of HIV-1 infection, with 5- to 16-fold greater activity than the wild-type molecule. The enhanced inhibitory activity by fusion-defective mutations correlates with local structural perturbations close to the cavity that destabilize the six-helix bundle. Taken together, these results indicate that the conserved hydrophobic coiled-coil cavity in the gp41 core is critical for HIV-1 entry and its inhibition and provides a potential antiviral drug target.

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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

Journal of Virology ◽

10.1128/jvi.74.1.326-333.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 326-333 ◽

Cited By ~ 109

Author(s):

Karl Salzwedel ◽

Erica D. Smith ◽

Barna Dey ◽

Edward A. Berger

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Target Cells ◽

Effector Cells ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Soluble Cd4

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.

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