HIV-1 Env-mediated Membrane Fusion Research Articles

Sphingomyelin Synthase 2, but Not Sphingomyelin Synthase 1, Is Involved in HIV-1 Envelope-mediated Membrane Fusion

Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.

Co-Expression of Foreign Proteins Tethered to HIV-1 Envelope Glycoprotein on the Cell Surface by Introducing an Intervening Second Membrane-Spanning Domain

The envelope glycoprotein (Env) of human immunodeficiency virus type I (HIV-1) mediates membrane fusion. To analyze the mechanism of HIV-1 Env-mediated membrane fusion, it is desirable to determine the expression level of Env on the cell surface. However, the quantification of Env by immunological staining is often hampered by the diversity of HIV-1 Env and limited availability of universal antibodies that recognize different Envs with equal efficiency. To overcome this problem, here we linked a tag protein called HaloTag at the C-terminus of HIV-1 Env. To relocate HaloTag to the cell surface, we introduced a second membrane-spanning domain (MSD) between Env and HaloTag. The MSD of transmembrane protease serine 11D, a type II transmembrane protein, successfully relocated HaloTag to the cell surface. The surface level of Env can be estimated indirectly by staining HaloTag with a specific membrane-impermeable fluorescent ligand. This tagging did not compromise the fusogenicity of Env drastically. Furthermore, fusogenicity of Env was preserved even after the labeling with the ligands. We have also found that an additional foreign peptide or protein such as C34 or neutralizing single-chain variable fragment (scFv) can be linked to the C-terminus of the HaloTag protein. Using these constructs, we were able to determine the required length of C34 and critical residues of neutralizing scFv for blocking membrane fusion, respectively.

Dissociation of the trimeric gp41 ectodomain at the lipid–water interface suggests an active role in HIV-1 Env-mediated membrane fusion

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.

GP41 Ectodomain Dissociates and Forms a Stable Monomer on Phospholipid Vesicles and Detergent Micelles: Implication for the HIV-1 Env-Mediated Membrane Fusion

The first step of HIV infection involves the fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoproteins, gp120 and gp41. The binding of gp120 to the cell surface receptors CD4 triggers a cascade of conformational changes that disrupt the gp41-gp120 interactions and allows the insertion of the N-terminal fusion peptide of gp41 into the host cell membrane. The gp41 trimer then rapidly folds into a 6-helix bundle that pulls together the fusion peptide, inserted in the host cell membrane and the transmembrane domain, located in the viral membrane. Very little structural information is known about the pre-fusion state of gp41 notwithstanding its critical importance for the design of fusion inhibitors. To investigate the dynamics and structural properties of such metastable states, we designed a set of protein constructs mimicking the extracellular ectodomain of gp41. We found that the secondary structure of gp41 ectodomain is pronouncedly impacted by the presence of phospholipid vesicles or detergents containing phosphatidyl choline head groups. Our NMR and multi-angle light scattering data attribute these changes to the transition between a trimer in the absence of detergent to a monomer in the presence of a membrane-mimicking environment. The structure of gp41 in a monomeric state was determined by state-of-the-art NMR techniques, including measurements of NOE distance restraints and residual dipolar couplings in weakly aligned solutions. 15N relaxation data provided a detailed view at the backbone dynamics of both the trimeric and monomeric conformations. This stable monomeric state may represent a crucial structural intermediate facilitating both the gp41 conformational change during fusion and the local apposition of the viral and cellular membranes.

Gelsolin activity controls efficient early HIV-1 infection

BackgroundHIV-1 entry into target lymphocytes requires the activity of actin adaptors that stabilize and reorganize cortical F-actin, like moesin and filamin-A. These alterations are necessary for the redistribution of CD4-CXCR4/CCR5 to one pole of the cell, a process that increases the probability of HIV-1 Envelope (Env)-CD4/co-receptor interactions and that generates the tension at the plasma membrane necessary to potentiate fusion pore formation, thereby favouring early HIV-1 infection. However, it remains unclear whether the dynamic processing of F-actin and the amount of cortical actin available during the initial virus-cell contact are required to such events.ResultsHere we show that gelsolin restructures cortical F-actin during HIV-1 Env-gp120-mediated signalling, without affecting cell-surface expression of receptors or viral co-receptor signalling. Remarkably, efficient HIV-1 Env-mediated membrane fusion and infection of permissive lymphocytes were impaired when gelsolin was either overexpressed or silenced, which led to a loss or gain of cortical actin, respectively. Indeed, HIV-1 Env-gp120-induced F-actin reorganization and viral receptor capping were impaired under these experimental conditions. Moreover, gelsolin knockdown promoted HIV-1 Env-gp120-mediated aberrant pseudopodia formation. These perturbed-actin events are responsible for the inhibition of early HIV-1 infection.ConclusionsFor the first time we provide evidence that through its severing of cortical actin, and by controlling the amount of actin available for reorganization during HIV-1 Env-mediated viral fusion, entry and infection, gelsolin can constitute a barrier that restricts HIV-1 infection of CD4+ lymphocytes in a pre-fusion step. These findings provide important insights into the complex molecular and actin-associated dynamics events that underlie early viral infection. Thus, we propose that gelsolin is a new factor that can limit HIV-1 infection acting at a pre-fusion step, and accordingly, cell-signals that regulate gelsolin expression and/or its actin-severing activity may be crucial to combat HIV-1 infection.

Biophysical property and broad anti-HIV activity of albuvirtide, a 3-maleimimidopropionic acid-modified peptide fusion inhibitor.

Albuvirtide (ABT) is a 3-maleimimidopropionic acid (MPA)-modified peptide HIV fusion inhibitor that can irreversibly conjugate to serum albumin. Previous studies demonstrated its in vivo long half-life and potent anti-HIV activity. Here, we focused to characterize its biophysical properties and evaluate its antiviral spectrum. In contrast to T20 (Enfuvirtide, Fuzeon), ABT was able to form a stable α-helical conformation with the target sequence and block the fusion-active six-helix bundle (6-HB) formation in a dominant-negative manner. It efficiently inhibited HIV-1 Env-mediated cell membrane fusion and virus entry. A large panel of 42 HIV-1 pseudoviruses with different genotypes were constructed and used for the antiviral evaluation. The results showed that ABT had potent inhibitory activity against the subtypes A, B and C that predominate the worldwide AIDS epidemics, and subtype B′, CRF07_BC and CRF01_AE recombinants that are currently circulating in China. Furthermore, ABT was also highly effective against HIV-1 variants resistant to T20. Taken together, our data indicate that the chemically modified peptide ABT can serve as an ideal HIV-1 fusion inhibitor.

Characterization of Antibody Responses Against the 2F5 Epitope ELDKWA sing HIV-1 Env-Mediated Membrane Fusion and Neutralization Assays

The epitope ELDKWA, which is located in the membrane-proximal external region (MPER) of HIV-1 gp41, is an important neutralizing epitope. The human monoclonal antibody (mAb) 2F5 against this epitope shows broad neutralizing activity toward many HIV strains. However, several reports have shown that the epitope-specific mAbs induced by peptides containing MPER did not exhibit the same neutralizing activities as human mAb 2F5. In this study, four ELDKWA epitope specific mAbs (9E7, 7E10, 6B5, and 2B4) induced by immunization with the ELDKWA epitope in varied molecular contexts, all showed inhibitory activities with different potencies in HIV-1 Env-mediated membrane fusion assays and pseudovirus neutralization assays. This result indicates that though these antibodies recognize the epitope ELDKWA, their characterizations differ from that of neutralizing antibodies, implying that the neutralizing mAbs can be induced but also need to be screened, and the protective ability of a related vaccine antigen depends on the concentration of the neutralizing mAbs in the induced polyclonal antibodies.

HIV-1 Env-Mediated Membrane Fusion Monitored by Fluorescence Microscopy of Syncytium Formation Between Clone69T1RevEnv and SupT1 Cells

Objectives: HIV-1 initiates infection by fusing with CD4+ lymphocytes or macrophages, mediated by the envelope protein Env (gp120/gp41). We developed a new HIV fusion assay using fluorescently labeled Clone69T1RevEnv cells expressing Env, and CD4+ SupT1 cells. We examined whether known inhibitors of HIV-1 infection, including peptides, lectins and antibodies inhibit membrane fusion in this system.Methods: Clone69TRevEnv (“HIV-Env”) cells were maintained in DMEM with heat-inactivated fetal bovine serum (FBS), L-glutamine, geneticin, hygromycin B and tetracycline. Env expression was induced by removing tetracycline. Cells were plated in 48-well plates for 24 h and labeled with Calcein-AM Green. SupT1 cells, maintained in RPMI 1640 with 10% FBS, penicillin, streptomycin and L-glutamine, were labeled with CellTrace(tm) Calcein red-orange. The SupT1 cells were incubated with adherent HIV-Env cells, with or without the inhibitors, for 3 h, and observed under a Nikon fluorescence microscope. Syncytia formation resulted in orange fluorescence.Results: Hippeastrum hybrid (Amaryllis) agglutinin (HHA), Galanthus nivalis (Snowdrop) agglutinin (GNA), and the peptide Enfuvirtide inhibited membrane fusion at 1 μg/ml. Anti-gp120 (IgG1B12, m14 IgG, and 2G12), and anti-gp41 (2F5 and 4E10) antibodies that inhibit HIV-1 infection, had little or no inhibitory effect on cell-cell fusion.Conclusion: Fluorescently labeled HIV-Env and SupT1 cells can be used to monitor HIV Env-mediated fusion as the fused cells change color. This assay can be adapted to screen novel inhibitors of membrane fusion in high-throughput assays. Our observation that antibodies that inhibit HIV infection do not inhibit syncytium formation, suggests that the mechanisms of cell-cell and virus-cell membrane fusion may be different. This observation also raises the possibility that the antibodies may not be able to inhibit the spread of viral genetic material from infected cells to uninfected cells.

The role of the N-terminal segment of CCR5 in HIV-1 Env-mediated membrane fusion and the mechanism of virus adaptation to CCR5 lacking this segment

BackgroundHIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4). The critical determinants of CCR5 coreceptor function are the N-terminal domain (Nt) and the second extracellular loop. However, mutations in gp120 adapt HIV-1 to grow on cells expressing the N-terminally truncated CCR5(Δ18) (Platt et al., J. Virol. 2005, 79: 4357–68).ResultsWe have functionally characterized the adapted Env (designated Env(NYP)) using a quantitative cell-cell fusion assay. The rate of fusion with target cells expressing wild-type CCR5 and the resistance to fusion inhibitors was virtually identical for wild-type Env and Env(NYP), implying that the coreceptor affinity had not increased as a result of adaptation. In contrast, Env(NYP)-induced fusion with cells expressing CCR5(Δ18) occurred at a slower rate and was extremely sensitive to the CCR5 binding inhibitor, Sch-C. Resistance to Sch-C drastically increased after pre-incubation of Env(NYP)- and CCR5(Δ18)-expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env(NYP)-CD4-CCR5(Δ18) complexes accumulate at sub-threshold temperature and that low-affinity interactions with the truncated coreceptor are sufficient for triggering conformational changes in the gp41 of Env(NYP) but not in wild-type Env. We also demonstrated that the ability of CCR5(Δ18) to support fusion and infection mediated by wild-type Env can be partially reconstituted in the presence of a synthetic sulfated peptide corresponding to the CCR5 Nt. Pre-incubation of wild-type Env- and CCR5(Δ18)-expressing cells with the sulfated peptide at sub-threshold temperature markedly increased the efficiency of fusion.ConclusionWe propose that, upon binding the Nt region of CCR5, wild-type Env acquires the ability to productively engage the extracellular loop(s) of CCR5 – an event that triggers gp41 refolding and membrane merger. The adaptive mutations in Env(NYP) enable it to more readily release its hold on gp41, even when it interacts weakly with a severely damaged coreceptor in the absence of the sulfopeptide.

θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation

Retrocyclin-1, a -defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 microm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Sphingolipids, cholesterol, and HIV-1: A paradigm in viral fusion

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37 degrees C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.

Monoclonal Antibodies Recognizing HIV-1 gp41 Could Inhibit Env-Mediated Syncytium Formation

Some monoclonal antibodies (mAbs) could inhibit infection by HIV-1. In this study, four mAbs against HIV-1 gp41 were prepared in mice. All four mAbs could bind to the recombinant soluble gp41 and recognize the native envelope glycoprotein gp160 expressed on the HIV-Env + CHO-WT cell in flow cytometry analysis. Interestingly, the results show that all four mAbs purified by affinity chromatography could inhibit HIV-1 Env-mediated membrane fusion (syncytium formation) by 40%-60% at 10 μg/mL, which implies potential inhibitory activities against HIV-1.

Fusion of HIV-1 envelope-expressing cells to human glomerular endothelial cells through an CXCR4-mediated mechanism.

A central question in the pathogenesis of HIV-associated thrombotic microangiopathic (HIV-TMA) lesions is whether the HIV-1 envelope glycoprotein (HIV-1 Env) can interact directly with human glomerular endothelial cells (HGECs) through specific HIV-1 co-receptors. The goal of this study was to determine whether cultured primary HGECs express significant levels of the major HIV-1 co-receptors CD4, CXCR4, and/or CCR5 to allow fusion interactions with HIV-1. The expression of CD4, CXCR-4 and CCR-5 was assessed in cultured HGECs by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry using specific antibodies. The HIV-1 Env-mediated membrane fusion of target glomerular cells was evaluated by a fluorescent dye transfer-based cell-cell fusion microscopic method. HGECs express CXCR4 mRNA and protein as determined by RT-PCR and immunostaining with phycoerythrin-conjugated anti-CXCR4 Mab 12G5. CD4 and CCR5 were not detected in HGECs, either by RT-PCR or by surface immunostaining with specific antibodies. Incubation of HGECs with cells expressing a CD4-independent envelope strain (HIV-1IIIB-8x) and the CD4-dependent envelope strain (HIV-1IIIB) resulted in transfer of fluorescent dyes of approximately 20% after 8-16 h incubation at 37 degrees C. Incubation in the presence of inhibitors (C34, which blocks six-helix bundle formation, and AMD3100, which interacts with CXCR4) reduced dye transfer by 60%-80%, confirming that the dye transfer was specific with respect to gp120-gp41-mediated fusion. Cultured primary HGECs express CXCR4 but not CD4 or CCR5. The ability of HGECs to promote fusion by a CD4-independent HIV-1 envelope glycoprotein suggests that these cells may become a potential direct target of certain HIV-1 isolates.

An inhibitor of glycosphingolipid metabolism blocks HIV-1 infection of primary T-cells.

HIV-1 uses CD4 and chemokine receptors to enter cells. However, other target membrane components may also be involved. This study examines the role of glycosphingolipids (GSL) in HIV-1 entry into primary lymphocytes and its modulation by an inhibitor of GSL biosynthesis. CD4 lymphocytes purified from normal or the p-group subtype individuals that were defective in Gb3 synthesis were treated with a GSL biosynthesis inhibitor, 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP). The PPMP-treated cells were tested for HIV-1 replication by measuring p24 antigen production for 7-14 days post-infection and for susceptibility to HIV-1 Env-mediated fusion monitored by a fluorescent dye transfer assay. The effects of PPMP treatment on HIV-1 binding to CD4 lymphocytes were also examined by measuring HIV-1 p24. CD4 lymphocytes from p donors that are devoid of Gb3, but have elevated levels of GM3 were highly susceptible to HIV-1 fusion/entry. Pre-treatment of primary human CD4 lymphocytes from normal or p-sub-group type with PPMP, significantly reduced HIV-1 replication with no change in CD4 and CXCR4 levels. Inhibition of HIV-1 infection was due to the block in HIV-1 Env-mediated plasma membrane fusion. Binding of HIV-1 to CD4 lymphocytes was not affected by PPMP treatment. Manipulation of glycosphingolipid metabolic pathways may alter susceptibility of CD4 lymphocytes to HIV-1 entry.

LFA-1 adhesion molecules are not involved in the early stages of HIV-1 env-mediated cell membrane fusion.

A recently developed sensitive assay to examine the early stages of HIV-1 env-mediated cell fusion is based on the redistribution of fluorescent dyes between membranes and cytoplasm of adjacent cells, monitored by fluorescence video microscopy. This assay demonstrated that membrane fusion can occur under conditions where no syncytia are formed. Fusion started earlier than syncytia formation and was not very sensitive to HIV-1 env+/CD4+ cell ratios. In the current study, this assay was used to determine the role of LFA-1 in HIV-1 env-mediated membrane fusion and syncytia formation. CD4- LFA-1- Epstein-Barr virus transformed lines from two leukocyte adhesion deficiency patients were infected with recombinant vaccinia expressing gp120/41 (HIV-IIIB), and cocultured with CD4+ subclones of the human T cell line CEM, which were generated by chemical mutagenesis and express either normal (LFA-1+), or low levels of LFA-1 (LFA-1lo). It was found that the LFA-1lo T-cell clone formed much smaller and fewer syncytia compared to the LFA-1+ subclones, but both clones fused equally well with the gp120/41 expressing LFA-1- B cells as monitored by redistribution of fluorescent dyes. Furthermore, monoclonal antibodies against the LFA-1 molecules reduced the number of syncytia formed but had no effect on membrane fusion. These findings demonstrate that the adhesion molecule LFA-1 does not play a crucial role in the early events of HIV-1 env-mediated cell membrane fusion, but may contribute to the later events leading to giant cell formation.